bluuebunny/crossref_metadata_2025 huggingface dataset:
Evidence that the Peripheral‐Type Benzodiazepine Receptor Ligand Ro 5–4864 Inhibits β‐Endorphin Release from AtT‐20 Cells by Blockade of Voltage‐Dependent Calcium Channels
Authors: Bisserbe J. C. , Patel J. , Eskay R. L.
Published: October 2006

Abstract The demonstrations that Ro 5–4864, a ligand selective for the peripheral‐type benzodiazepine (BZD) binding site, inhibited cellular differentiation and proliferation and that occupancy of the peripheral‐type BZD binding site likely mediated the observed BZD effects on diverse endocrine tissues suggested that Ro 5–4864 disrupted a common cellular regulatory event. Using a well‐characterized anterior pituitary‐derived tumor cell line (AtT‐20 cells), which synthesizes and secretes adrenocorticotropic hormone (ACTH), β‐lipotropin hormone (β‐LPH), and β‐endorphin (BE), we have investigated the molecular mechanism of action of Ro 5–4864's capacity to alter BE secretion. Ro 5–4864 inhibits basal and induced BE release from AtT‐20 cells, through a cyclic AMP‐independent mechanism. Ro 5–4864 completely blocked the corticotropin‐releasing hormone and forskolin‐induced release of BE without altering the concomitant production of cyclic AMP. The addition to AtT‐20 cells of CGP 28392, a dihydropyridine that has been demonstrated in other systems to specifically activate by increasing concentrations of Ro 5–4864. In contrast to the capacity of Ro 5–4864 to block CGP‐induced BE release, Ro 5–4864 lacked the capacity to block enhanced BE secretion due to the calcium ionophore A23187, which increases intracellular Ca2+ levels independent of the voltage‐dependent Ca2+ channels. Our findings suggest that Ro 5–4864 inhibits BE secretion from AtT‐20 cells through a blockade of the voltage‐dependent membrane Ca2+ channels and this mechanism of action may be responsible for Ro 5–4864's diverse effects observed on other cell types.
Is Dopamine‐Induced Inhibition of Adenylate Cyclase Involved in the Autoreceptor‐Mediated Negative Control of Tyrosine Hydroxylase in Striatal Dopaminergic Terminals?
Authors: El Mestikawy S. , Hamon M.
Published: October 2006

Abstract The mechanism of the negative control of tyrosine hydroxylase (TH) activity induced by the stimulation of presynaptic 3,4‐dihydroxyphenylethylamine (dopamine, DA) autoreceptors was investigated using rat striatal slices and synaptosomes incubated under control ([K+] = 4.8 mM) or depolarizing ([K+] = 60 mM) conditions. The stimulation of DA autoreceptors by 7‐hydroxy‐2‐(di‐n‐propylamino) tetralin (1 μM 7‐OH‐DPAT) produced a significant decrease in TH activity extracted from striatal slices maintained under control conditions. This effect was associated with the complete conversion of TH into an enzyme form with a low affinity for its pterin cofactor (Km∼0.80 mM). Furthermore, compared to TH extracted from control tissues, that from 7‐OH‐DPAT‐exposed striatal slices was more sensitive to the stimulatdry effects of exogenous heparin and cyclic AMP‐dependent phosphorylation. Such changes were opposite to those induced by incubating striatal slices with the adenylate cyclase activator forskolin. Indeed, forskolin treatment completely converted TH into an enzyme form with a high affinity for its pterin cofactor (Km∼0.16 mM). Such conversion was associated with a shift in the optimal pH for TH activity from 5.8 (control) to 7.2 (forskolin). Under depolarizing conditions, the blockade by (—)‐sulpiride of the stimulation of DA autoreceptors by endogenous DA was associated with a marked activation of TH. Modifications of enzymatic characteristics triggered by (—)‐sulpiride were then similar to those induced by forskolin treatment. These data suggest that presynaptic DA autoreceptors modulate the activity of TH by controlling the degree of cyclic AMP‐dependent phosphorylation of the enzyme. The blockade by Pertussis toxin of the 7‐OH‐DPAT‐induced inhibition of TH activity is coherent with a possible negative coupling of presynaptic DA autoreceptors (closely related to the D2 type) with adenylate cyclase. Such negative coupling would account for the reduction of TH activity when presynaptic DA autoreceptors are stimulated.
Decreased Transcription of Neuronal Polyadenylated RNA During Senescence in Nuclei from Rat Brain Cortex
Authors: Dan Lindholm B.
Published: October 2006

Abstract Neuronal and glial cell‐enriched nuclei were prepared from the brain cortex of rats of different ages for study of alterations in the synthesis of cellular RNA with age. RNA synthesis by isolated neuronal nuclei was substantially reduced in senescent rats, whereas that of glial nuclei remained constant throughout development. The nuclear content of polyadenylic acid‐containing RNA in neurons was particularly decreased in old rats. The results show that the activity of the chromatin‐bound neuronal RNA polym‐erase declines during senescence, an observation indicating an age‐related reduction in template activity in neuronal nuclei. The activity of nuclear poly(adenylate) polymerase also progressively decreases in aging neurons. The decrease in the transcription and polyadenylation of nuclear RNA may contribute to the decline in neuronal protein synthesis observed in old animals.
The Brain 68‐Kilodalton Microtubule‐Associated Protein Is a Cognate Form of the 70‐Kilodalton Mammalian Heat‐Shock Protein and Is Present as a Specific Isoform in Synaptosomal Membranes
Authors: Whatley S. A. , Leung T. , Hall C. , Lim L.
Published: October 2006

Abstract The relationship between the 68‐kilodalton microtubule‐associated protein (68KMAP) and the major heat‐induced protein (HSP70) in rat and human cells was investigated by comparison of their heat induction properties and by tryptic and Cleveland peptide mapping procedures. HSP70 synthesis was induced by heat shock of rat and human cells, whereas 68KMAP was a major synthesised protein in the absence of heat shock, with its synthesis being only slightly increased on heat shock. Tryptic peptide mapping, however, indicated strong peptide homology between the two proteins. These data, therefore, confirm that 68KMAP represents a constitutively expressed, heat‐shock cognate gene. Two‐dimensional gel electrophoretic analysis of subcellular fractions of rat brain, combined with peptide mapping procedures, indicated that 68KMAP exists as at least two isoforms separable by isofocussing, the more acidic of which (α68KMAP) is present in fractions enriched in microtubules, cytosol, microsomes, synaptosomal plasma membranes, and synaptic vesicles, and the more basic of which (β68KMAP) is present predominantly in fractions enriched in synaptic vesicles and synaptosomal plasma membranes. These two forms are distinguishable in terms of changes in Cleveland peptide maps, and we conclude that α‐ and β68KMAP, therefore, represent distinct forms. The significance of these findings to the molecular pathogenesis of Down's syndrome in the human brain is discussed.
Protein Kinase C‐Activating Tumor Promoters Enhance the Differentiation of Astrocytes in Aggregating Fetal Brain Cell Cultures
Authors: Honegger Paul
Published: October 2006

Abstract: Serum‐free aggregating cell cultures of fetal rat telencephalon treated with the potent tumor promoter phorbol 12‐myristate 13‐acetate (PMA) showed a marked, rapid, and sustained increase in the activity of the astrocyte‐specific enzyme glutamine synthetase (GS). This effect was accompanied by a small increase in RNA synthesis and a progressive reduction in DNA synthesis. Only mitotically active cultures were responsive to PMA treatments. Since in aggregate cultures astrocytes are the preponderant cell type, both in number and mitotic activity, it can be concluded that PMA induces and/or enhances the terminal differentiation of astrocytes. The developmental expression of GS was also greatly stimulated by mezerein, a potent nonphorbol tumor promoter, but not by 4α‐phorbol 12,13‐didecanoate, a nonpromoting phorbol ester. Since both tumor promoters, PMA and mezerein, are potent and specific activators of C‐kinase, it is suggested that C‐kinase plays a regulatory role in the growth and differentiation of normal astrocytes.
Splanchnic Nerve Transection Abolishes the Age‐Dependent Increase of Neuropeptide Y‐Like Immunoreactivity in Rat Adrenal Gland
Authors: Higuchi Hiroshi , Yang Hsiu‐Ying T.
Published: October 2006

Abstract: Using an antiserum directed against porcine neuropeptide Y (NPY), a high concentration of NPY immunoreactivity (NPY‐IR) was detected in rat adrenal gland. The level of NPY‐IR in the adrenal gland was found to increase considerably with age. Biochemical characterization by reverse‐phase HPLC indicated that this increase was due to accumulations of NPY and another molecular form of NPY‐like immunoreactive peptide. Chronic denervation of the splanchnic nerve abolished this age‐dependent increase of NPY‐IR in rat adrenal gland.
Ontogenetic Profile of the Adenosine Uptake Sites in Rat Forebrain
Authors: Morgan P. F. , Montgomery P. , Marangos P. J.
Published: October 2006

Abstract: The ontogenesis of rat forebrain adenosine uptake sites labelled by [3H]nitrobenzylthioinosine ([3H]NBI) was determined and compared to that of rat forebrain adenosine receptors labelled by N6‐cyclohexyl[3H]adenosine ([3H]‐CHA). [3H]NBI binding is highly invariant with similar levels of [3H]NBI binding sites from embryonic day 19 to day 30 postpartum. Scatchard and Hill analyses reveal the binding of [3H]NBI in 6‐day‐old tissue to be indistinguishable from such binding in 30‐day‐old tissue. In contrast, [3H]‐CHA binding is highly variant. [3H]CHA binding develops slowly but steadily from about embryonic day 19, with adult binding levels being achieved at around 25 days postpartum. The ontogenetic profile of [3H]CHA appears to coincide with synaptogenesis whereas that of [3H]NBI does not.
Neonatal and Adult 6‐Hydroxydopamine‐Induced Lesions Differentially Alter Tachykinin and Enkephalin Gene Expression
Authors: Sivam Subbiah P. , Breese George R. , Krause James E. , Napier T. Celeste , Mueller Robert A. , Hong Jau‐Shyong
Published: October 2006

Abstract: The present investigation examined the effects of neonatal and adult 6‐hydroxydopamine (6‐OHDA)‐induced lesions of dopaminergic neurons on opioid and tachykinin peptides and their gene expression in the rat basal ganglia. This work was undertaken to determine if changes in these neuropeptide systems were contributing to the differing behavioral responses observed between neonatally and adult‐lesioned rats after dopamine agonist administration. [Met5]Enkephalin (ME) content was increased in striatal tissue from both 6‐OHDA‐lesioned groups when compared with unlesioned controls. Dynorphin‐A (1–8) content was not altered by the 6‐OHDA lesions. The tachykinin peptides substance P and neurokinin A were significantly decreased in level in the striatum and substantia nigra of neonatally lesioned rats, but not in the adult‐lesioned rats, when compared with unlesioned controls. Proenkephalin mRNA abundance (quantified by an RNA‐cDNA hybridization technique) and precursor level (as reflected by cryptic ME content) were increased in the striatum of both neonatally and adult‐lesioned rats. The abundance of preprotachykinin mRNA coding for the tachykinin peptides was markedly decreased in the neonatally lesioned rats, whereas only a small reduction was observed in the adult‐lesioned rats. These results suggest that destruction of dopamine‐containing terminals with 6‐OHDA elevates the level of ME by accelerating transcriptional and/or translational processes; conversely, the reduced content of tachykinins in neonatally lesioned rats may be due to a reduction in such processes. Thus, preproenkephalin‐A and preprotachykinin gene expression are differentially regulated after lesioning of catecholamine‐containing neurons, an observation suggesting a close functional relationship among these neurotransmitter systems. Furthermore, of the peptides studied, only levels of the tachykinin peptides were differentially altered in the striatum and substantia nigra of the neonatally lesioned rats compared with adult‐lesioned rats; therefore, these peptides may be associated with the distinctive behavioral differences between neonatally and adult 6‐OHDA‐lesioned rats given dopamine agonists.
A Phorbol Ester‐Sensitive Kinase Catalyzes the Phosphorylation of P0 Glycoprotein in Myelin
Authors: Brunden Kurt R. , Poduslo Joseph F.
Published: October 2006

The proposed structural protein of peripheral nerve myelin, P0, has been shown to have several covalent modifications. In addition to being glycosylated, sulfated, and acylated, P0 is phosphorylated, with the intracellular site of this latter addition being in question. By employing nerve injury models that exhibit different levels of P0 biosynthesis in the absence and presence of myelin assembly, we have examined the cellular location of P0 phosphorylation. It is demonstrated that there is comparable P0 phosphorylation in both normal and crush‐injured adult rat sciatic nerves, although the level of biosynthesis of P0 differs between these myelin maintaining and actively myelinating nerve models, respectively. The glycoprotein does not appear to be phosphorylated readily in the transected adult sciatic nerve, a preparation in which P0 biosynthesis is observed but that lacks myelin membrane. These observations suggest that the modification is not associated with the biosynthesis or maturation of P0 in the endoplasmic reticulum or Golgi, but that it instead occurs after myelin assembly. That P0 phosphorylation occurs in the normal nerve even when translation is inhibited by cycloheximide treatment lends further support to this conclusion. P0 is shown to be phosphorylated on one or more serine residues, with all or most of the phosphate group(s) being labile as evidenced by pulse‐chase analysis. Addition of a biologically active phorbol ester, 12‐O‐tetradecanoylphorbol‐13‐acetate or 4β‐phorbol 12, 13‐dibutyrate, substantially increases the extent of [32P]orthophosphate incorporation into the glycoprotein of normal and crushed nerve but not transected nerve. Biologically inactive 4α‐phorbol 12, 13‐didecanoate has no effect on P0 phosphorylation. Similarly, the addition of the cyclic AMP analog 8‐bromo‐cyclic AMP causes no appreciable changes in P0 labeling. These findings indicate that the phorbol ester‐sensitive enzyme, protein kinase C, may be responsible for the phosphorylation of P0 within the myelin membrane.
Developmental and Functional Studies of Parvalbumin and Calbindin D28K in Hypothalamic Neurons Grown in Serum‐Free Medium
Authors: Pfyffer Gaby E. , Faivre‐Bauman Annie , Tixier‐Vidal Andrée , Norman Anthony W. , Heizmann Claus W.
Published: October 2006

The Ca2+‐binding proteins parvalbumin (Mr= 12K) and calbindin D28K [previously designated vitamin D‐dependent Ca2+‐binding protein (Mr= 28K)] are neuronal markers, but their functional roles in mammalian brain are unknown. The expression of these two proteins was studied by immunocytochemical methods in serum‐free cultures of hypothalamic cells from 16‐day‐old fetal mice. Parvalbumin is first detected in all immature neurons, but during differentiation, the number of parvalbumin‐immunoreactive neurons greatly declines to a level reminiscent of that observed in vivo, where only a subpopulation of neurons stains for parvalbumin. In contrast, calbindin D28K was expressed throughout the period investigated only in a distinct subpopulation of neurons. Depolarization of fully differentiated hypothalamic neurons in culture resulted in a dramatic decrease of parvalbumin immunoreactivity but not of calbindin D28K immunoreactivity. The parvalbumin staining was restored on repolarization. Because the anti‐parvalbumin serum seems to recognize only the metal‐bound form of parvalbumin, the loss of immunoreactivity may signal a release of Ca2+ from intracellular parvalbumin during depolarization of the cells. We suggest that parvalbumin might be involved in Ca2+‐dependent processes associated with neurotransmitter release.
Two‐Site Immunoassay for Acetylcholinesterase in Brain, Nerve, and Muscle
Authors: Brimijoin S. , Hammond P. , Rakonczay Z.
Published: October 2006

Two‐site methods were developed for immunoassay of acetylcholinesterase (AChE; EC 3.1.1.7) in crude extracts of rat and human tissues. A radiometric assay for human AChE utilized a specific monoclonal AChE antibody adsorbed to polystyrene microtiter wells at alkaline pH. AChE bound strongly to this antibody after 24 h at 4°C. Bound enzyme was detected with an 125I‐labeled antibody against a different AChE epitope. The assay signal was quasilinearly related to AChE concentration in purified and crude samples, with a detection threshold near 100 pg. Tetrameric and dimeric AChE behaved equivalently in the assay. Two‐site methods with a different pair of species‐selective antibodies worked equally well for immunoassay of rat AChE. Assays of the rat enzyme showed that immunoreactivity was lost as rapidly as enzyme activity during heating to 54°C. On the other hand, immunoreactivity was preserved despite loss of enzyme activity after exposure to anti cholinesterases or trypsin. A biotinylated second antibody detected by alkaline‐phosphatase‐conjugated avidin was used to develop an AChE enzyme‐linked immunosorbent assay (ELISA) with a sensitivity similar to that of the radio‐metric assay. Either the ELISA or the radiometric immunoassay may be useful whenever proteolysis or other mechanisms are suspected of dissociating enzyme activity and immunoreactivity. In denervated muscle and ligated peripheral nerve, application of the two‐site method showed closely parallel variations in immunoreactivity and enzyme activity.
Effects of Substance P on Nicotinic Acetylcholine Receptor Function in PC 12 Cells
Authors: Simasko Steven M. , Durkin Jule A. , Weiland Gregory A.
Published: October 2006

Abstract: The effects of substance P on the functioning of nicotinic acetylcholine receptors in PC12 cells were examined. Carbachol‐stimulated 22Na+ uptake was used to assess the functional state of the nicotinic receptor. We found that incubation of the cells with substance P alone caused a loss of receptor function. Receptors recovered from this effect with a t1/2 of 0.94 ± 0.10 min. Since receptors recovered from carbachol‐induced desensitization at a significantly slower rate (t1/2, 1.77 ± 0.21 min), it was concluded that the two inactive states are not kinetically equivalent. The effects of substance P on carbachol‐induced loss of receptor activity were also examined. Substance P had no effect on a component of carbachol‐induced loss of activity that was non re‐coverable (inactivation). However, substance P had several effects on the recoverable loss of activity induced by carbachol (desensitization). Substance P caused a shift to the left in the EC50 for carbachol‐induced desensitization at equilibrium. If cells were simultaneously incubated with carbachol and substance P7‐11, a low‐potency analog of substance P, an increase in the rate of formation of a state of the receptor that was kinetically indistinguishable from the state induced by carbachol alone was observed. However, not all inhibition of nicotinic cholinergic function could be explained by an increased rate of formation of a desensitized receptor and it is concluded that substance P causes both enhanced desensitization and block of the nicotinic receptor‐linked channel.
Taurine Biosynthesis in Rat Brain In Vivo: Lack of Relationship with Cysteine Sulfinate Decarboxylase Glutamate Decarboxylase‐Associated Activity (GAD/CSDII)
Authors: Legay Frangois , Lecestre Dominique , Tappaz Marcel
Published: October 2006

Abstract: Two distinct forms of cysteine sulfinate decarboxylase (CSD), respectively, CSDI and CSDII, have already been separated in rat brain. One of them, CSDII, appeared to be closely associated wtih glutamate decarboxylase (GAD). We have investigated whether the taurine concentration in brain was dependent on CSDII activity in vivo. CSDI and CSDII activities were specifically measured in crude brain extracts after selective immunotrapping. After 4 days of chronic treatment of mice with γ‐acetylenic γ‐aminobutyric acid, a drastic and identical decrease in CSDII and GAD activities was observed in the brain. Taurine concentration and CSDI activities were not significantly altered. Following striato‐nigral pathway lesioning in the rat brain, GAD and CSDII show an identical 80% decrease in the substantia nigra. In contrast, CSDI activity and taurine concentration in the substantia nigra were similarly but only slightly affected with an about 30% decrease. Our results provide further evidence that GAD and CSDII are indeed the same enzyme. They show that CSDII does not play any role in the biosynthesis of taurine in vivo. Our findings suggest that CSDI might be the biosynthetic enzyme for taurine in vivo and that there might be some endings projecting into the substantia nigra that contain CSDI and taurine.
Binding of [3H]SKF 38393 to Dopamine D‐l Receptors in Rat Striatum In Vitro; Estimation of Receptor Molecular Size by Radiation Inactivation
Authors: Gredal Ole , Nielsen Mogens
Published: October 2006

Abstract: [3H]SKF 38393 (2,3,4,5,‐tetrahydro‐7,8‐dihy‐droxy‐l‐phenyl‐lH‐3‐benzazepine) binds with high affinity to 3,4‐dihydroxyphenylethylamine (dopamine) D‐l receptors in rat striatum in vitro (KD= 7 and 14 nM in nonfrozen and frozen striatum, respectively). The number of binding sites (5max) was ∼ 80.0 pmol/g of original tissue, a value similar to the Bmax for the dopamine D‐l antagonist SCH 23390. Nondisplaceable [3H]SKF 38393 binding was ∼45% of total binding. Irradiation (0–4 Mrad) of frozen whole striata decreased the number of [3H]SKF 38393 binding sites monoexponentially without changing the binding affinity. The functional molecular mass for the agonist dopamine D‐1 binding site was 132,800 daltons, which is higher than the functional molecular mass of the antagonist dopamine D‐1 binding site (∼80,000 daltons).
Regulation of Dipeptidyl Aminopeptidase I and Angiotensin Converting Enzyme Activities in Cultured Murine Brain Cells by Cortisol and Thyroid Hormone
Authors: Davis Michael H. , Pieringer Joanne
Published: October 2006

Abstract: Cultures of dissociated brain cells from 15‐day‐old fetal mice were grown in the presence and absence of 20 or 50 nM triiodothyronine (T3), 30 or 300 nM cortisol, and 30 nM cortisol plus 50 nM T3 added to chemically defined media or in media supplemented with 15% serum from control and hypothyroid calves. The specific activities of five lysosomal enzymes—N‐acetyl galactosaminidase, β‐gluc‐uronidase, β‐galactosidase, cathepsin B, and dipeptidyl aminopeptidase I (DAP‐I)—were higher in cells grown in calf serum than in cells grown in defined media. Of these enzymes, only DAP‐I was elevated in activity when the cells were grown in hypothyroid calf serum instead of control calf serum. Elevation of DAP‐I activity was reversed by addition of 20 nM T3 to hypothyroid calf serum. The enzymatic properties of DAP‐I were similar whether the cells were grown in control or hypothyroid calf serum and were similar to those reported for human fibroblasts and the purified enzyme. When the cells were grown in defined media, cortisol decreased the activities of all lysosomal enzymes, with 300 nM cortisol being more effective than 30 nM cortisol. Addition of 50 nM T3 to 30 nM cortisol decreased DAP‐I activity more than 30 nM cortisol alone, but 50 nMT3 alone in defined media did not alter DAP‐I levels. The reduction of DAP‐I activity in these cells by T3 required cortisol, unidentified components in serum, or both. All enzyme activities increased during the period when these cells normally make myelin membrane components. Significant amounts of all lysosomal enzymes were found in the defined media except cathepsin B and DAP‐I. These cells also contained an angiotensin converting enzyme (ACE) activity that was chloride dependent, inhibited by 2 μM angiotensin converting enzyme inhibitor, and inactivated by 1 mM EDTA. ACE levels were increased in cells exposed to 50 nM T3, 30 nM cortisol, and 50 nM T3 plus 30 nM cortisol. Significant amounts of ACE activity were also excreted by these cells into defined media.
Neuromelanogenic and Cytotoxic Properties of Canine Brainstem Peroxidase
Authors: Grisham Matthew B. , Perez Vernon J. , Everse Johannes
Published: October 2006

Abstract: We have isolated a heme protein from canine midbrains that possesses potent peroxidase activity. This enzyme catalyzes the oxidation of dopamine to neuromela‐nin in the presence of H2O2. We have further shown that the isolated peroxidase possesses potent cytotoxic activity in the presence of superoxide or H2O2 and Cl−. The enzyme possesses an endogenous NAD(P)H oxidase activity that can promote the cytotoxic activity by virtue of its production of superoxide. Other enzymes such as dihydroorotate dehydrogenase and galactose oxidase, which produce O2−and H2O2, respectively, are also effective in promoting the cytotoxic activity of the brainstem peroxidase. Although rat erythrocytes were routinely used as the target cell, other cell types, including rat hepatoma and mouse neuroblastoma cells, are also susceptible to the toxic action of the peroxidase. The cytotoxic action of the brainstem peroxidase is dramatically enhanced by kainic acid and is significantly enhanced by Mn2+, whereas dopamine was found to be a potent inhibitor of the cytotoxic activity. Based on these findings, we postulate a central role for the brainstem peroxidase in dopamine metabolism as well as in the biochemical and anatomical changes associated with Parkinson's disease.
Acetylcholine Releases Prostaglandins from Brain Slices Incubated In Vitro
Authors: Reichman Melvin , Nen Wu , Hokin Lowell E.
Published: October 2006

Abstract: A variety of neurotransmitters elicit a phos‐phoinositide response in the CNS; however, their effects on prostaglandin (PG) formation in the brain are not well characterized. In the present study, we investigated the effect of acetylcholine (ACh) on the synthesis of PCs E and F in slices from various regions of guinea pig brain incubated in glucose‐fortified Krebs‐Henseleit bicarbonate saline. Slices were prewashed in the presence of 1% albumin to reduce basal PG levels followed by incubation for 30 min at 37°C in the presence or absence of ACh. Under these conditions, 5 mM ACh significantly increased the efflux of PGE and PGF from brain regions enriched in muscarinic cholinergic receptors, i.e., cerebral cortex, temporal cortex, corpus striatum, and hippocampus. Depolarization by 45 mM KCl also significantly enhanced PG synthesis, and the relative magnitude of the effect was similar to that of ACh. The stimulation of PG synthesis by ACh was inhibited by 20 μM atropine, whereas the K+‐induced stimulation was not. The effects of potassium and ACh were additive at maximally effective ACh concentrations, an observation that suggests that ACh and K+ increase PG efflux through independent mechanisms. Norepinephrine, histamine, and serotonin, three other neurotransmitters that evoke a phos‐phoinositide response in the brain, were ineffective in stimulating PG release from brain cortex slices.
Characteristics of Partially Purified Nerve Growth Factor Receptor
Authors: Stach Robert W. , Lyons C. Richard , Perez‐Polo J. Regino
Published: October 2006

Abstract: Receptors for the nerve growth factor protein (NGF) have been isolated from three cell types [embryonic chicken sensory neurons (dorsal root sensory ganglia; DRG), rat pheochromocytoma (PC 12) and human neuroblastoma (LAN‐1) cells] and have been shown to be similar with respect to equilibrium dissociation constants. The present results demonstrate that there are multiple molecular weight species for NGF receptors from DRG neurons and PC 12 cells. NGF receptors can be isolated from DRG as four different molecular species of 228, 187, 125, and 112 kilodaltons, and PC 12 cells as three molecular species of 203, 118, and 107 kilodaltons. The NGF receptors isolated from DRG show different pH‐binding profiles for high‐and low‐affinity binding. High‐affinity binding displays a bell‐shaped pH profile with maximum binding between pH 7.0 and 7.9, whereas low‐affinity binding is constant between pH 5.0 and 9.1, with a twofold greater binding at pH 3.6. At 22°C, the association rate constant was found to be 9.5 ± 1.0 ± 106M‐1 S‐1. Two dissociation rate constants were observed. The fast dissociating receptor has a dissociation rate constant of 3.0 ± 1.5 ± 10‐2 S‐1, whereas the slow dissociating receptor constant was 2.4 ± 1.0 ± 10‐4S‐1. The equilibrium dissociation constants calculated from the ratio of dissociation to association rate constants are 2.5 ± 10‐11M for the high‐affinity receptor (type I) and 3.2 ± 10‐9M for the low‐affinity receptor (type II). These values are the same as those determined by equilibrium experiments on the isolated receptors. The data are consistent with the hypothesis that there are at least two different receptors for NGF.
Insulin Binding and Effects on Pyrimidine Nucleoside Uptake and Incorporation in Cultured Mouse Astrocytes
Authors: Kum W. , Cockram C. S. , Zhu S. Q. , Teoh R. , Vallance‐Owen J. , Young J. D.
Published: October 2006

Abstract: Binding of 125I‐insulin to primary cultures of differentiated mouse astrocytes was time‐dependent, reaching equilibrium after 2 h at 22°C, with equilibrium binding corresponding to 20.79 fmol/mg of protein, representing ∼5,000 occupied binding sites/cell. The half‐life of 125I‐insulin dissociation at 22°C was 2 min, with an initial dissociation rate constant of 4.12 ± 10‐2 s‐1. Dissociation of bound 125I‐insulin was not accelerated significantly in the presence of unlabeled insulin (16.7 μM). Porcine and desoctapeptide insulins competed for specific 125I‐insulin binding in a dose‐dependent manner, whereas growth hormone, glucagon, and somatostatin did not. For porcine insulin, Scatchard analysis suggested multiple‐affinity binding sites (high‐affinity Ka= 4.92 ± 108M‐1; low‐affinity Ka= 0.95 ± 107M‐1). After incubation with insulin (0.5 μM) for 2 h at 37°C, increases above basal values of 254 ± 23 and 189 ± 34% for [3H]uridine uptake and incorporation, respectively, were observed. After incubation with insulin (0.5 μM) for 24 h at 37°C, there were increases of 145 ± 6% for [3H]thymidine uptake and 166 ± 11% for thymidine incorporation. Basal and stimulated undine and thymidine uptake and incorporation were inhibited by 50 μM dipyridamole. These studies confirm that mouse astrocytes in vitro possess specific insulin receptors and demonstrate an effect of insulin on pyrimidine nucleoside uptake and incorporation.
Effect of Increased Glucose Levels on Na+/K+‐Pump Activity in Cultured Neuroblastoma Cells
Authors: Yorek Mark A. , Dunlap Joyce A. , Ginsberg Barry H.
Published: October 2006

Abstract: Neuroblastoma cells were used to analyze the effect of elevated glucose levels on myo‐inositol metabolism and Na+/K+‐pump activity. The activity of the Na+/K+ pump in neuroblastoma cells is almost totally sensitive to ouabain inhibition. Culturing neuroblastoma cells in 30 mM glucose caused a significant decrease in Na+/K+‐pump activity, myo‐inositol metabolism, and myo‐inositol content, compared to cells grown in the presence of 30 mM fructose. Glucose supplementation also caused a large intracellular accumulation of sorbitol. The aldose reductase inhibitor sorbinil prevented the abnormalities in myo‐inositol metabolism and partially restored Na+/K+‐pump activity in neuroblastoma cells cultured in the presence of elevated glucose levels. These results suggest that the accumulation of sorbitol by neuroblastoma cells exposed to elevated concentrations of extracellular glucose causes a decrease in myo‐inositol metabolism and these abnormalities are associated with a reduction in Na+/K+‐pump activity.
Stimulation of Formation of Inositol Phosphates in Primary Cultures of Bovine Adrenal Chromaffin Cells by Angiotensin II, Histamine, Bradykinin, and Carbachol
Authors: Plevin Robin , Boarder Michael R.
Published: October 2006

Abstract: Histamine, bradykinin, and angiotensin II stimulate release of catecholamines from adrenal medulla. Here we show, using bovine adrenal chromaffin cells in culture, that these agonists as well as carbachol (with hexamethonium) stimulate production of inositol phosphates. The histamine response was mepyramine sensitive, implicating an H1 receptor, whereas bradykinin had a lower EC50 than Met‐Lys‐bradykinin, and [Des‐Arg9]‐bradykinin was relatively inactive, implicating a BK‐2 receptor. Total inositol phosphates formed in the presence of lithium were measured, with histamine giving the largest response. The relative contribution of chromaffin cells and nonchromaffin cells in the responses was assessed. In each case chromaffin cells were found to be responding to the agonists; in the case of histamine the response was solely on chromaffin cells. When the inositol phosphates accumulating over 2 or 5 min, with no lithium present, were separated on Dowex anion‐exchange columns, bradykinin gave the greatest stimulation in the inositol trisphosphate fraction, whereas histamine gave a larger inositol monophosphate accumulation. On resolution of the isomers of stimulated inositol trisphosphate after 2 min of stimulation, the principal isomer present was inositol 1,3,4‐trisphosphate in each case. Two hypotheses for the differential responses to histamine and bradykinin are discussed.
Multiple Human Tyrosine Hydroxylase Enzymes, Generated Through Alternative Splicing, Have Different Specific Activities in Xenopus Oocytes
Authors: Horellou P. , Bourdellès B. Le , Clot‐Humbert J. , Guibert B. , Leviel V. , Mallet J.
Published: October 2006

Abstract: A single human tyrosine hydroxylase (HTH) gene has been shown previously to generate four species of mRNA by alternative splicing. The four different HTH mRNAs were independently synthesized in vitro, using the SP6 transcription system. Each of these mRNA species was able to direct the synthesis of an active form of TH following injection into Xenopus oocytes. Quantitation of synthesized HTH polypeptides allowed the determination of the relative specific activity of each individual HTH form. A significant difference in specific activity was found between each form, suggesting that alternative splicing may play a role in regulating HTH activity in vivo.
Regional Cerebral Glucose Metabolism and Blood Flow During the Silent Phase of Methylmercury Neurotoxicity in Rats
Authors: Hargreaves Richard J. , Eley Brenda P. , Moorhouse Stephen R. , Pelling David
Published: October 2006

Abstract: Methylmercuric chloride was given to rats in a neurotoxic dose regimen (six daily doses of 8 mg kg−1 p.o.). During the silent (asymptomatic) phase of intoxication, the rates of cerebral glucose influx and cerebral glucose phos‐phorylation were measured simultaneously using 2‐deoxy‐glucose. Regional cerebral blood flow was also measured using iodoantipyrine. The unidirectional flux of glucose into brain was not affected by methylmercury, and differences in the rates of glucose phosphorylation from region to region remained coupled to the regional cerebral blood flow. However, the blood flow was reduced throughout the brain, an observation suggesting that the operational level of metabolically regulated blood flow had been reset. Thus, in spite of a generalised reduction in blood flow, there was no indication of impaired cerebral glucose supply or utilization during the silent phase of methylmercury intoxication.
Modulation of the Electrically Evoked Release of 5‐[3H]Hydroxytryptamine from Rat Cerebral Cortex: Effects of Alpidem, CL 218872, and Diazepam
Authors: Lista Alvaro , Arbilla Sonia , Langer Salomon Z.
Published: October 2006

Abstract: The effect of ω (benzodiazepine)‐receptor agonists, antagonists, and inverse agonists on the electrically evoked release of 5‐[3H]hydroxytryptamine ([3H]5‐HT) was studied in superfused slices of the rat frontal cerebral cortex. The electrically evoked release of [3H]5‐HT was enhanced by nanomolar concentrations of diazepam and the selective oM1‐receptor agonists alpidem and CL 218872. The ω/ω2‐ and ω1‐receptor antagonists flumazenil and CGS 8216, respectively, did not modify the electrically evoked release of [3H]5‐HT. The ω1‐receptor agonist Ro 5–4864 and the ω1‐receptor inverse agonist ethyl‐β‐carboline‐3‐carboxylate on their own did not affect the electrically evoked release of [3H]5‐HT. On the other hand, the inverse agonist 6,7‐dimethoxy‐4‐ethyl‐β‐carboline‐3‐carboxylic acid methyl ester (DMCM), at micromolar concentrations, inhibited both the spontaneous and the evoked release of [3H]5‐HT. The facilitation of the electrically evoked release of [3H]5‐HT by diazepam, alpidem, or CL 218872 was potentiated by γ‐aminobutyric acid (GABA). Exposure to flumazenil and CGS 8216 antagonized the facilitation by diazepam, alpidem, or CL 218872 of [3H]5‐HT release. The inhibition of the release of [3H]5‐HT by DMCM was not modified by exposure to either flumazenil, CGS 8216, or GABA. The inhibitory effect of DMCM was not observed when monoamine oxidase activity was inhibited by pargyline. Our results indicate that activation of ω1‐re‐ceptors by agonists facilitates the electrically evoked release of [3H]5‐HT from slices of the rat frontal cerebral cortex, and that these ω1‐receptors appear to be associated in a macromolecular complex with the GABA receptor. Inverse agonists acting on the ω1‐GABA receptor complex are not involved in the modulation of the release of [3H]5‐HT.
Polyamines Differentially Inhibit Cyclic AMP‐Dependent Protein Kinase‐Mediated Phosphorylation in the Brain of the Tobacco Hornworm, Manduca sexta
Authors: Combest Wendell L. , Bloom Timothy J. , Gilbert Lawrence I.
Published: October 2006

Abstract: The effects of the naturally occurring polyamines spermine and spermidine on phosphorylation promoted by cyclic AMP (cAMP)‐dependent protein kinase (PK) (cAMP‐PK; EC 2.7.1.37) were studied using the brain of the tobacco hornworm, Manduca sexta. Four particulate‐associated peptides (280, 34, 21, and 19 kilodaltons) in day 1 pupal brains are endogenous substrates for a particulate type II cAMP‐PK. These phosphoproteins are present in brain synaptosomal, as well as microsomal, particulate fractions but are not present in the cytosol. They are distributed throughout the CNS and PNS and are present in several nonneuronal tissues as well. Phosphorylation of these proteins via cAMP‐PK was inhibited markedly by micromolar concentrations of spermine and spermidine. Other particulate‐associated peptides phosphorylated via a Ca2+/calmodulin‐PK or Ca2+ and cAMP‐independent PKs were unaffected by polyamines, whereas the phosphorylation of a 260–kilodalton peptide was markedly enhanced. Spermine did not exert its inhibitory effect indirectly by enhancement of cAMP or ATP hydrolysis or via proteolysis, but its action appears to involve a substrate‐directed inhibition of cAMP‐PK‐promoted phosphorylation as well as enhanced dephosphorylation. Although addition of spermine resulted in marked ribosome aggregation in synaptosomal and microsomal particulate fractions, this phenomenon was not involved in the inhibition of cAMP‐PK‐promoted phosphorylation.
Astrocytes from Forebrain, Cerebellum, and Spinal Cord Differ in Their Responses to Vasoactive Intestinal Peptide
Authors: Cholewinski A. J. , Wilkin G. P.
Published: October 2006

Abstract: Astrocytes from cortex, cerebellum, and spinal cord responded to isoproterenol and vasoactive intestinal peptide (VIP) with increases in intracellular cyclic AMP levels. The response to VIP was as great as that to isoproterenol in cortical astrocytes (180–fold and 185–fold, respectively), and the effect of VIP in combination with isoproterenol was partially additive. Spinal cord astrocytes also responded to VIP and isoproterenol with equal potency (seven‐ to ninefold and eight‐to 13–fold, respectively), but the level of response was much smaller than in cortex. Spinal cord astrocytes were synergistic in their response to VIP and isoproterenol. The response to VIP was lowest in cerebellar astrocytes (only threefold), and no additivity was observed when VIP was added together with isoproterenol. A small response to α‐melanocyte stimulating hormone (α‐MSH) was also observed in cortex and cerebellum, but not in spinal cord. Somatostatin inhibited the response to isoproterenol in cortex and cerebellum, but had no effect in spinal cord. The results from the above study show that astrocytes obtained from these three regions of the rat CNS express quite different responses to NIP and α‐MSH and further point to possible astrocyte heterogeneity.
Ontogeny of the GTP‐Binding Protein Go in Rat Brain and Heart
Authors: Asano Tomiko , Kamiya Nobuko , Semba Reiji , Kato Kanefusa
Published: October 2006

Abstract: We determined the ontogeny of the GTP‐binding protein Go in rat brain and heart by employing highly sensitive enzyme immunoassay methods. In the brain, the α subunit of Go (Goα) gradually increased and reached adult levels approximately 20 and 30 days after birth in cerebral cortex and cerebellum, respectively. Concentrations of β subunits, which were also quantified by the immunoassay, were almost equal to those of Goα in the brain of rats younger than 10 days, but were higher than those of Goα after 10 days. These results suggest the late development of GTP‐binding proteins other than Go. Goα was immunohistochemically positive in neuropils and negative in cell bodies at any age tested. In the heart, the concentrations of Goα increased up to several times of the adult level just after birth, and then gradually decreased after the 20th postnatal day. The level of Goα in the liver, however, was very low and constant throughout ontogenic development. An immunohistochemical study indicated that Goα was positive in the cardiac muscle of young rat, but negative in that of adult rat. These results indicate that Goα exists in cells other than those of nervous tissues and neuroendocrine cells in some periods of ontogenic development.
Effects of Extracellular Potassium on Glycogen Stores of Astrocytes In Vitro
Authors: Cambray‐Deakin Martin , Pearce Brian , Morrow Christine , Murphy Sean
Published: October 2006

Abstract: Astrocyte‐enriched and meningeal cell cultures of the rat cerebral cortex were prepared, and their glycogen content was measured after 10–90 min under control (2.5 mM) concentrations of potassium after prefeeding with 20 mM glucose. No net change in glycogen level was noted in either culture over this period. Cell cultures were then exposed to increased concentrations of potassium (5, 10, and 15 mM), and their glycogen content was measured after 10–90 min. Both types of cell culture showed complex and variable changes in glycogen content. In general, increased potassium concentrations caused astrocyte glycogen stores to be reduced at physiological increases of potassium levels (from 2.5 to 5 mM and above), although a period of resynthesis was evident at all potassium concentrations. Meningeal cell glycogen levels were highly variable and only affected by high (10 and 15 mM) levels of potassium. These results are discussed with respect to the theory that changes in the external potassium concentration caused by neuronal activity might act as a signal controlling astrocyte glycogen stores.
Foot‐Shock Stress and Anxiogenic β‐Carbolines Increase t‐[35S]Butylbicyclophosphorothionate Binding in the Rat Cerebral Cortex, an Effect Opposite to Anxiolytics and γ‐Aminobutyric Acid Mimetics
Authors: Concas A. , Serra M. , Atsoggiu T. , Biggio G.
Published: October 2006

Abstract: The effect of foot‐shock stress on t‐[35S]‐butylbicyclophosphorothionate ([35S]TBPS) binding to fresh unwashed membrane preparations from rat cerebral cortex was studied and was compared to those of GABAA receptor agonists and antagonists and to positive and negative modulators of the GABAergic transmission. [35S]TBPS binding was increased in the cerebral cortex of rats exposed to foot shock compared to that of nonstressed rats. Scatchard analysis revealed that the effect of foot shock was due to an increase in the total number of [35S]TBPS binding sites. In contrast, the in vitro addition of muscimol or GABA induced a dose‐dependent inhibition of [35S]TBPS binding, an effect abolished by the concomitant addition of the GABA receptor antagonist, bicuculline, which, per se, enhanced [35S]TBPS binding by 73%. Thus, bicuculline, similar to stress, increased [35S]TBPS binding in the same membrane preparation. In contrast to stress, the anxiolytic and positive modulators of the GABAergic transmission (ZK 93423, ZK 91296, and di‐azepam) inhibited the specific binding of [35S]TBPS in a concentration‐dependent manner. The greatest inhibitory effect was produced by ZK 93423 at 30 μM (31% of control), followed by diazepam (54% of control) and by the partial agonist ZK 91296 (61 % of control). Scatchard plot analysis indicated that the inhibition induced by ZK 93423 and diazepam was due to a decrease in the density of [35S]TBPS recognition sites. On the other hand, the anxiogenic β‐carbolines DMCM and FG 7142 mimicked the effect of stress. Thus, at a 10 μf concentration, DMCM and FG 7142 increased [35S]TBPS binding by 22% and 26%, respectively. The inhibitory effect of ZK 93423, ZK 91296, and diazepam and the stimulatory effect of FG 7142 and DMCM were blocked by the benzo‐diazepine receptor antagonists Ro 15–1788 and ZK 93426. The demonstration that stress, similar to the GABA receptor antagonist bicuculline and to anxiogenic β‐carbolines and opposite to GABA agonists, anxiolytic β‐carbolines, and benzodiazepines, increases [35S]TBPS binding in the rat cerebral cortex, suggests that some emotional state related to stress and anxiety may result from a diminished GABAergic transmission at the level of the GABAA/benzodiazepine receptor/chloride ionophore complex.
Galanin‐Like Immunoreactivity Is Unchanged in Alzheimer's Disease and Parkinson's Disease Dementia Cerebral Cortex
Authors: Beal M. Flint , Clevens Ross A. , Chattha Geetinder K. , MacGarvey Usha M. , Mazurek Michael F. , Gabriel Steven M.
Published: October 2006

Abstract: Galanin is a recently isolated neuropeptide that is of particular interest in dementing disorders because of its known colocalization with choline acetyltransferase in magnocellular neurons of the basal nucleus of Meynert. These neurons degenerate in Alzheimer's disease, and there is a corresponding deficiency of cortical choline acetyltransferase activity. In the present study, galanin‐like immunoreactivity was measured in the postmortem cerebral cortex and hippocampus of 10 controls and 14 patients who had had Alzheimer's disease. Significant reductions of choline acetyltransferase activity (50–60%) were found in all regions examined; however, there was no significant effect on concentrations of galanin‐like immunoreactivity. Similar measurements were made in postmortem tissues of 12 control and 13 demented Parkinsonian patients who had had Alzheimer‐type cortical pathology. Choline acetyltransferase activity was again significantly decreased in all regions examined but there were no significant reductions in galanin‐like immunoreactivity. Experimental lesions of the fornix in rats produced parallel significantly correlated reductions of both choline acetyltransferase activity and galanin‐like immunoreactivity in the hippocampus. Galanin‐like immunoreactivity in the human hypothalamus consisted of two molecular‐weight species on gel‐permeation chromatography, and two forms were resolved by reverse‐phase HPLC. The paradoxical preservation of galanin‐like immunoreactivity, despite depletion of the activity of choline acetyltransferase, with which it is colocalized, is as yet unexplained. Recent studies have shown that galanin inhibits both acetylcholine release in the hippocampus and memory acquisition; therefore, preserved galanin may exacerbate the cholinergic and cognitive deficits that accompany dementia.