bluuebunny/crossref_metadata_2025_split huggingface dataset:
Transport of Asparagine by Rat Brain Synaptosomes: An Approach to Evaluate Glutamine Accumulation
Authors: Erecińska Maria , Zaleska Malgorzata M. , Chiu Linda , Nelson David 
Published: October 2006

AbstractIsolated rat brain synaptosomes accumulated L‐asparagine with a Km value of 348 μM and a Kmax value of 3.7 nmol/mg of protein/min at 28°C. Uptake of L‐asparagine was inhibited by the presence of L‐glutamine, whereas transport of L‐glutamine was blocked by L‐asparagine. Alanine, serine, cysteine, threonine, and, in particular, leucine were also inhibitory whereas α‐(methylamino)isobutyrate, ornithine, lysine, arginine, and glutamate were much less effective blockers. Transport of L‐asparagine had a substantial sodium‐dependent component, whereas that of the D‐stereoisomer was almost unaffected by the presence or absence of the cation. L‐Asparaginc was accumulated to a maximal gradient, [L‐Asn]i/[L‐Asn]o, of 20–30, and this value was reduced to 5–6 by withdrawal of sodium or addition of high [KG]. A plot of log [Na+]o/{Na+]i against the log [L‐Asn]i/[L‐Asn]o had a slope close to 1, which indicates that a single sodium ion is transported inward with each asparagine molecule. It is postulated that uptake of L‐asparagine occurs, to a large extent, in cotransport with Na+ and that it utilizes the sodium chemical gradient and the membrane electrical potential as the source of energy. The similarity between the L‐asparagine and L‐glutamine transport systems and the reciprocal inhibition of influx of the two amino acids suggest that the same mechanism is responsible for glutamine accumulation. This could explain the high [GIn]i maintained by the brain in vivo.
Activation and Inactivation of Oxytocin and Vasopressin Release from Isolated Nerve Endings (Neurosecretosomes) of the Rat Neurohypophysis
Authors: Payza Kemal , Russell James T. 
Published: October 2006

AbstractNeurosecretory terminals (neurosecretosomes, NSS) were isolated from rat neurohypophyses. High [K+]oor veratridine stimulated secretion of vasopressin and oxytocin by up to ∼ 100‐fold. Stimulated secretion was dependent on calcium and temperature, and could be elicited from NSS maintained in culture for 4 days. After overnight culture of the NSS, secretion was still inhibited by calcium channel blockers (cobalt, dihydropyridines, ω‐conotoxin, D 600) and K opiates (dynorphin and U50488). Ionomycin evoked dose and calcium‐dependent hormone release, with a Hill coefficient for calcium of 1.74. High [K+]o enhanced the 5 μMionomycin‐induced secretion, apparently through calcium entry rather than depolarization, as the increase in secretion was abolished by 100 μM D 600. During prolonged depolarization the hormone secretion peaked within 2 min, then declined to near basal levels. Depolarization for 25 min without calcium neither activated secretion nor prevented subsequent secretion on readdition of calcium, suggesting that the decline in secretion was not due to membrane depolarization. Indeed, the rates of decline in secretion were similar for different levels of depolarization (0.070 ± 0.003 and 0.081 ± 0.003 min−1 for 25 and 45 mM [K+]o, respectively). Four minutes after the onset of continuous depolarization (45 mM[K+]o) in the presence of calcium, the declining secretion was still dependent on voltage‐activated calcium influx through channels sensitive to D 600 and nitrendipine. The results presented here suggest that the decline in secretion during prolonged depolarizing stimuli may be due to exhaustion, inactivation, or desensitization of a calcium‐triggered event.
Galactose‐1‐Phosphatase in Rat Brain
Authors: Gulavita Srima J. , Zhang Li‐Ping , Dougherty John J. , Dain Joel A. 
Published: October 2006

AbstractA prominent galactose‐1‐phosphatase was isolated from rat brain and partially purified by chromatography on diethylaminoethyl‐Sephacel, hydroxylapatite, and Sephacryl S‐300 columns. The galactose‐1‐phosphatase was separated from alkaline phosphatase, and from two forms of glucose‐1‐phosphatase. The three columns gave a 10‐fold increase in specific activity to 290 mol/min/mg of protein, with a yield of 15%. Of the eight sugar phosphates tested, galactose‐1‐phosphate was the best substrate for the purified enzyme, followed by glucose‐1‐phosphate, which was hydrolyzed 40% as rapidly as galactose‐1‐phosphate. Galactose‐1‐phosphatase had an optimum pH of 8.5 and a Km value of 2.5 mM for galactose‐1‐phosphate hydrolysis. Mg2+ was required for activity, and supported half‐maximal activity at a concentration of 1.25 mM. Phosphate was the only potent inhibitor found. ATP, arsenate, and vanadate caused moderate inhibition of 10 mM levels, whereas AMP, L‐homoarginine, and L‐phenylalanine stimulated enzyme activity. Galactose‐1‐phosphatase was determined to have a Stokes radius of 30 A and a sedimentation coefficient of 4.IS. These values were used to calculate a molecular weight of 50,200 and a factional ratio showing the enzyme to be a globular protein. It is hypothesized that a similar phosphatase may play a role in reducing brain galactose‐1 ‐phosphate concentrations in patients with galactosemia.
Tyrosine Hydroxylase mRNA Expression by Dopaminergic Neurons in Culture: Effect of 1 ‐Methyl‐4‐Phenylpyridinium Treatment
Authors: Beck Klaus D. , Knusel Beat , Pasinetti Giulio , Michel Patrick P. , Zawadzka Halina , Goldstein Menek , Hefti Franz 
Published: October 2006

AbstractTo enable us to study expression of tyrosine hydroxylase [TH; tyrosine 3‐monooxygenase; L‐tyrosine tetrahydropteridine: oxygen oxidoreductase (3‐hydroxylating); EC 1.14.16.2] as a measure of dopaminergic neuron function in future experiments, methods were developed to quantify TH mRNA levels in cultures of dopaminergic mesencephalic cells. The model of selective dopaminergic toxicity of 1‐methyl‐4‐phenylpyridinium (MPP+) was used to verify the specificity of our methods. Fetal (embryonic day 15) rat ventral mesencephalic cell cultures were treated with 15 μMMPP+ for 48 h, conditions previously shown to reduce the number of TH‐immunoreactive neurons, TH activity, and dopamine uptake to 5–10% of control values. This treatment decreased the number of neurons labeled by TH in situ hybridization to 9% of untreated controls and caused a strong reduction of the abundance of TH mRNA in Northern blots. Our findings establish TH mRNA expression as a parameter for future studies of toxic and trophic effects on cultured dopaminergic neurons, and they support the view that MPP+ destroys dopaminergic neurons.
Iminodipropionitrile‐Induced Dyskinesia in Mice: Striatal Calcium Channel Changes and Sensitivity to Calcium Channel Antagonists
Authors: Bangalore Ramesh , Hawthorn Mark , Triggle David J. 
Published: October 2006

AbstractAdministration of 3, 3′‐iminodipropionitrile (IDPN) (1g/kg, i.p. for 3 days) in mice leads to the development of a characteristic syndrome consisting of lateral and vertical head and neck movements, hyperactivity, random circling, increased locomotor activity, and increased startle response. Nifedipine, verapamil, and diltiazem (10 mg/kg) inhibited significantly the symptoms of IDPN‐induced dyskinesia. However, there was no change in the affinity (KD) or the density of PN 200–110 binding sites (Bmax) in whole brains of IDPN‐treated mice. Similarly, the K+‐depolarization‐dependent Ca2+ uptake in synaptosomes from whole brain, cortex, or striatum was not altered following IDPN treatment. However, IDPN caused a significant increase in the Bmax value (from 157 ± 7 fmol/mg to 237 ± 31 fmol/mg in control and treated groups, respectively) of PN 200–110 binding to the striatum without change of KD value (38 ± 4.7 pM versus 33 ± 1.6 pM). IDPN also caused a slight but significant decrease in the KD value (from 68 ± 10.1 pM to 45 ± 4.5 pM in control and treated groups, respectively), without significant change of Bmax value (563 ± 51 fmol/mg versus 485 ± 41 fmol/mg) of PN 200–110 binding to the cortex. IDPN did not alter ω‐conotoxin binding in whole brain, striatum, or cortex. The behavioral effects of chronic IDPN treatment are inhibited by L‐type calcium channel antagonists and this may be associated with the observed increase in striatal L‐type calcium channels.
Multiple Neurite Formation in Neuroblastoma Cell Lines by Griseolic Acid, a Potent Inhibitor of Cyclic Nucleotide Phosphodiesterases
Authors: Mitsui Kazuhiko , Tsuji Shuichi , Yamazaki Mitsuo , Nagai Yoshitaka 
Published: October 2006

AbstractThe morphological change of several neuroblastoma cell lines induced by griseolic acid, a novel and potent inhibitor of cyclic nucleotide phosphodiesterase (PDE), was examined. In the cell lines tested, Neuro‐2a (a murine neuroblastoma cell line) showed dose‐dependent (1 μM‐1 mM) neurite extension. Griseolic acid markedly increased the intracellular cyclic AMP level of Neuro‐2a cells, suppressed DNA synthesis (82% at 1 mM), and induced multipolar (multiple‐neurite‐bearing)‐type neuritogenesis. A similar type of neurite outgrowth was induce by 8‐bromo‐cyclic AMP, which also elevated the intracellular cyclic AMP concentration. In contrast, when Neuro‐2a cells were treated with retinoic acid, neurite formation was of the monopolar (single‐neurite‐bearing) type. Papaverine and theophylline, which have been frequently used as PDE inhibitors, failed to induce these morphological changes up to 1 mM, probably owing to the lesser potency of these compounds as compared with griseolic acid on the inhibition of PDE. Retinoic acid, theophylline, and papaverine were ineffective at elevating the intracellular cyclic AMP level. These results suggest that multipolar‐type cell shape change in Neuro‐2a cells is correlated with the accumulation of intracellular cyclic AMP and that griseolic acid is a useful compound to induce neuroblastoma cells into terminal differentiation.
Nerve Growth Factor Potentiates Bradykinin‐Induced Calcium Influx and Release in PC12 Cells
Authors: Bush Andrew B. , Borden Laurence A. , Greene Lloyd A. , Maxfield Frederick R. 
Published: October 2006

AbstractTo investigate how the response to agonists changes during neuronal differentiation, we examined the effect of nerve growth factor (NGF) on bradykinin‐induced calcium increases in PC12 cells. Short‐term (1h) treatment with NGF increased the potency of bradykinin to raise intracellular calcium by about 10‐fold, whereas long‐term (1 week) treatment, which was associated with the expression of the differentiated phenotype, increased the potency about 100‐fold. Neither treatment affected the maximal response to bradykinin. NGF alone had no acute effect on calcium levels. Short‐term potentiation appeared to be mainly a result of greater release of calcium from intracellular stores, whereas the effect of long‐term treatment apparently was due to increases in both release from intracellular stores and calcium influx. [3H]Bradykinin binding to intact PC12 cells was unaltered by short‐term NGF treatment, whereas differentiated cells displayed a 50% increase in receptor number and about a twofold increase in affinity as compared with cells not treated with NGF. The production of inositol phosphates in response to bradykinin correlated poorly with the calcium transients, in that large calcium responses were associated with small increases in inositol phosphates. Neither NGF treatment had a significant effect on the appearance of inositol phosphates in response to bradykinin. Experiments with permeabilized cells revealed that differentiated cells did not display a heightened response to exogenously added inositol 1, 4, 5‐trisphosphate. Our results demonstrate that NGF modulates the bradykinin signaling pathway without acutely activating this pathway itself.
Presence of Calmodulin and Calmodulin‐Binding Proteins in the Nuclei of Brain Cells
Authors: Vendrell Montserrat , Aligué Rosa , Bachs Oriol , Serratosa Joan 
Published: October 2006

AbstractThe nuclear calmodulin levels have been measured in rat neurons and glial cells. The values are 1.0 and 1.1 γg/ mg of protein, respectively. These levels are about threefold higher than those in the nuclei of rat liver cells. We have also investigated the presence of several calmodulin‐binding proteins in the nuclei of both brain cellular types. As similarly observed in the nuclei of liver cells, we detected the presence of a‐spectrin and a 62‐kDa calmodulin‐binding protein (p62) in the nuclei of neurons and glial cells by irnmunoblotting and immunocytochemical methods. Both proteins are enriched in the purified nuclear matrix samples from both cellular types. In contrast to that occurring in rat hepatocytes, we have not been able to detect, by irnmunoblotting methods, caldesmon in the nuclear matrices of neurons and glial cells. The immunocytochemical studies suggest, however, that caldesmon can be present in the nuclei but in a fraction distinct from the nuclear matrices.
Stimulatory and Inhibitory Effects of N‐Methyl‐D‐Aspartate on 3H‐Inositol Polyphosphate Accumulation in Rat Cortical Slices
Authors: Baird John G. , Nahorski Stefan R. 
Published: October 2006

AbstractThe actions of the excitatory amino acid N‐methyl‐D‐aspartate (NMDA) on the accumulation of 3H‐inositol polyphosphate isomers in rat cerebral cortex slices have been examined over short (less than 5 min) incubation periods. NMDA caused the dose‐dependent accumulation of only [3HJinositol monophosphate and [3H]inositol bisphosphate (maximal effect between 0.3 and 1 mM), with no increase in [3H]inositol trisphosphate ([3H]InsP3) and (3H]inositol tetrakisphosphate ([3H]InsP4). HPLC analysis confirmed this, showing no increases in the breakdown products of [3H]Ins(1, 3, 4, 5)P4. When present with the muscarinic agonist carbachol (1 mM), high concentrations of NMDA (1 mM) could almost totally inhibit carbachol‐induced accumulation of 3H‐inositol polyphosphates. In contrast, at lower concentrations of NMDA (10 γM), the inhibitory effect was replaced with a synergistic accumulation of inositol polyphosphates, especially [3H]InsP4 and [3H]InsP3. The inhibitory effects of NMDA were only apparent when extracellular Ca2+ was present, although incubation in media with no added Ca2+resulted in somewhat reduced stimulatory responses to NMDA alone, but suppressed totally the inhibitory effects of 1 mM NMDA and reduced the synergistic effects of 10 γMNMDA on carbachol responses. These studies, therefore, reveal Ca2+‐dependent effects of NMDA indicative of indirect mechanisms of action and show that care must be made in interpreting the effects of NMDA on phosphoinositide metabolism unless the inositol polyphosphate composition has been fully characterised.
Modulation of Proteolytic Activity During Neuritogenesis in the PC12 Nerve Cell: Differential Control of Plasminogen Activator and Plasminogen Activator Inhibitor Activities by Nerve Growth Factor and Dibutyryl‐Cyclic AMP
Authors: Leprince P. , Register B. , Delrée P. , Rigo J. M. , Andre B. , Moonen G. 
Published: October 2006

AbstractExtracellular proteolysis is considered to be required during neuritic outgrowth to control the adhesiveness between the growing neurite membrane and extracellular matrix proteins. In this work, PC12 nerve cells were used to study the modulation of proteolytic activity during neuronal differentiation. PC12 cells were found to contain and release a 70–75‐kDa tissue‐type plasminogen activator (tPA) and a much less abundant 48‐kDa urokinase‐type plasminogen activator. A plasminogen activator inhibitor (PAI) activity with molecular sizes of 54 and 58 kDa was also detected in PC12 cell conditioned medium and formed high‐molecular‐mass complexes with released tPA. Release of PAI activity was dependent on treatment with nerve growth factor (NGF), whereas tPA synthesis and release were under control of a cyclic AMP‐ dependent mechanism and increased on treatment with dibutyryl‐cyclic AMP [(But)2cAMP] or cholera toxin. Simultaneous treatment with NGF and (But)2cAMP resulted in increases of both tPA and PAI release and enhancement of tPA‐PAI complex formation. The resulting plasminogen activator activity in conditioned medium was high in (But)2cAMP‐treated cultures with short neuritic outgrowth but remained low in NGF‐ or NGF plus (But)2 cAMP‐treated cultures, where neurite extension was, respectively, large and very large. These results suggest that excess proteolytic activity may be detrimental to neuritic outgrowth and that not only PAI release but also tPA‐PAI complex formation is associated with production of large and stable neuritic outgrowth. This can be understood as an involvement of PAI in the protection against neurite‐destabilizing proteolytic activity.
Guanine Nucleotides Regulate 2‐[125I]Iodomelatonin Binding Sites in Chick Retinal Pigment Epithelium but Not in Neuronal Retina
Authors: Chong Nelson W. S. , Sugden David 
Published: October 2006

AbstractThe characteristics of the binding sites labeled by the radioligand 2‐[125I]iodomelatonin were compared in chicken neuronal retina and retinal pigment epithelium (RPE). Specific binding of 2‐[125I]iodomelatonin in both sites was stable, saturable, reversible, and of high affinity. Scatchard analysis revealed an affinity constant (KD) of 446 ± 55 pMand a total number of binding sites (8max) of 25.4 ± 2.2 fmol/ mg of protein for neuronal retina. For RPE the KD was 34.1 ±2.2 pM and the Bmax 59.5 ± 5.2 fmol/mg of protein. Competition experiments with various melatonin analogues gave the following order of affinities: 2‐iodomelatonin < 2‐chloromelatonin > melatonin > 6‐chloromelatonin > 6‐hydroxymelatonin > N‐acetylserotonin > 6‐methoxyharmalan > 5‐hydroxytryptamine. Linear regression of log Kivalues from neuronal retina and RPE gave a highly significant dorrelation (r =0.994, n = 8; p <0.001). GTP inhibited specific binding to RPE membranes in a concentration‐dependent manner, but not in neuronal retinal membranes. The present results strongly suggest that a single type of melatonin receptor is found in neuronal retina and RPE, and that the site in RPE is coupled to a guanine nucleotide‐binding regulatory protein (G protein), but that in neuronal retina is not.
Solubilization and Partial Characterization of Angiotensin II Receptors from Rat Brain
Authors: Siemens I. R. , Swanson G. N. , Fluharty S. J. , Harding J. W. 
Published: October 2006

AbstractRat brain angiotensin II (Ang II) receptors were solubilized with a yield of 30–40% using the synthetic detergent 3[(3‐cholamidopropyl)dimethylammonio)]‐1 ‐propane‐sulfonate. Kinetic analysis employing the high‐affinity antagonist I25I‐Sar1, lle8‐Ang II indicated that the solubilized receptors exhibited the same properties as receptors present within intact brain membranes. Furthermore, there was a positive correlation (r −0.99) between the respective pIC50values of a series of agonist and antagonists competing for 125I‐Sarl,IIe8‐Ang II labeled binding sites in either solubilized or intact membranes. Moreover, covalent labeling of 125I‐ Ang II to solubilized receptors with the homo‐bifunctional cross‐linker disuccinimidyl suberate, followed by gel filtration, revealed one major and one minor binding peak with apparent molecular weights of 64,000 and 115,000, respectively. Two binding proteins of comparable molecular weights (i.e., 112,000 and 60,000) were also identified by covalent cross‐linking of I25l‐Ang II to solubilized brain membranes followed by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis analysis. In contrast, only the smaller molecular mass binding protein was observed when solubilized membranes were labeled with the antagonist 125I‐Sar1.IIe8‐Ang II prior to gel filtration, and chromatofocusing of antagonist labeled sites revealed only one peak with an isodectric point of 6.2. The successful solubilization of these binding sites should facilitate continued investigation of Ang II receptors in the brain.
Angiotensin II Increases Catecholamine Release from Bovine Adrenal Medulla but Does Not Enhance That Evoked by K+ Depolarization or by Carbachol
Authors: Powis D. A. , O'Brien K. J. 
Published: October 2006

Abstract: The effect of angiotensin II on catecholamine release from bovine adrenal medulla has been investigated. In retrogradely perfused, isolated bovine adrenal glands, angiotensin II increased basal efflux of catecholamines, but the presence of angiotensin II did not increase the release of catecholamines evoked either by bolus injections of the secretogogue carbachol or by depolarization with a perfusing solution containing a raised concentration of K+. In chromaffin cells maintained in primary tissue culture, angiotensin II increased 3H release from cells preloaded with [3H]‐noradrenaline but did not enhance the release evoked by carbachol or by depolarization with K+. The increase in 3H release evoked by angiotensin II from chromaffin cells in tissue culture was inhibited by its analogue antagonist Sar1,Ala8‐angiotensin II (saralasin) and was entirely dependent on the presence of Ca2+ in the experimental medium. These findings suggest that, in the chromaffin cells of the bovine adrenal medulla, angiotensin II acts on specific receptors to cause a calcium‐dependent catecholamine release but triggers no additional response that acts synergistically with depolarizing or nicotinic stimuli to augment catecholamine release.
Age‐Dependent Changes in the Subcellular Distribution of Rat Brain μ‐Opioid Receptors and GTP Binding Regulatory Proteins
Authors: Bem Wojciech T. , Yeung S. Jim , Belcheva Mariana , Barg Jacob , Coscia Carmine J. 
Published: October 2006

Abstract: The relative subcellular distributions of μ‐opioid receptors and guanine nucleotide binding regulatory proteins (G proteins) in 1‐day‐old (P1) and adult rat forebrain were compared. Light membranes (LMs) were resolved from heavy membranes (HM) by sucrose density gradient centrifugation. Marker enzyme analyses indicated that LMs contained most of the endoplasmic reticulum and Golgi complexes, whereas HMs were enriched in plasma membranes. Binding distribution and properties of μ‐opioid sites were assessed using [3H][d‐Ala2,Me‐Phe4,Gly‐ol5]enkephalin. P1 LMs possessed 43% of die total μ‐opioid binding detected compared to 16% in the adult. Although NaCl inhibited μ binding in LMs to a greater extent than in HMs, age‐dependent differences were not observed. P1 LM μ binding possessed greater sensitivity to 5′‐guanylylimidodiphosphate than their adult counterpart. Moreover, P1 LMs contained more Gocr. protein than P1 HMs or adult LMs, as demonstrated by immunoblotting with antisera against Gocr after one‐or two‐dimensional gel electrophoresis. These results suggest that P1 LMs contain a greater proportion of newly synthesized intracellular μ sites than adult LMs.