bluuebunny/crossref_metadata_2025 huggingface dataset:
Histamine Increases Phospholipid Methylation and H2‐Receptor‐Adenylate Cyclase Coupling in Rat Brain
Authors: Ozawa Koichiro , Segawa Tomio
Published: October 2006

Abstract: Histamine stimulated the enzymatic synthesis of phosphatidylcholine from phosphatidylethanolamine in crude synaptic membranes of rat brain containing the methyl donor S‐adenosyl‐l‐methionine (SAM). In the presence of, but not in the absence of SAM, histamine increased cyclic AMP accumulation at the concentrations that stimulate phospholipid methylation. S‐Adenosyl‐l‐homocysteine, an inhibitor of phospholipid methyltransferases, inhibited histamine‐stimulated phospholipid methylation and histamine‐induced cyclic AMP accumulation in the presence of SAM in a concentration‐dependent manner. Histamine‐induced [3H]methyl incorporation into phospholipids exhibited a marked regional heterogeneity in rat brain in the order of cortex > medulla oblongata > hippocampus > striatum > midbrain > hypothalamus. The regional distribution of histamine‐induced cyclic AMP accumulation exactly paralleled histamine‐stimulated [3H]‐methyl incorporation in rat brain. Histamine‐induced cyclic AMP accumulation was inhibited by the addition of cimetidine or famotidine, but not by mepyramine or diphenhydramine. The accumulation of cyclic AMP in the presence of SAM was observed by the addition of impromidine or dimaprit, but not by 2‐pyridylethylamine. These results indicate that phospholipid methylation is induced by histamine and may participate in H2‐receptor‐mediated stimulation of adenylate cyclase in rat brain.
Induction of Rat Brain Tubulin Following Ammonium Ingestion
Authors: Felipo Vicente , Miñana Maria‐Dolores , Azorín Inmaculada , Grisolía Santiago
Published: October 2006

Abstract: The effect of oral administration of ammonium acetate for 2, 15, 30, and 100 days on protein synthesis in rat brain was investigated. Although protein synthesis changes were modest, i.e., maximal increase of 24%, there was induction of synthesis and accumulation of a protein with an Mr of 55,000. We show, on the basis of its position on two‐dimensional electrophoresis and its immunological reactivity, that this protein is tubulin. Its content increased by 33% as determined by isolation of tubulin after 15 days of oral administration of ammonium and to 49% after 100 days as determined by quantitative immunoblotting.
Extracellular Amino Acid Concentrations in the Dorsal Spinal Cord of Freely Moving Rats Following Veratridine and Nociceptive Stimulation
Authors: Skilling Stephen R. , Smullin David H. , Beitz Alvin J. , Larson Alice A.
Published: October 2006

In vivo microdialysis was used to sample extracellular concentrations of amino acids in the dorsal lumbar spinal cord of freely moving rats. Changes in the extracellular concentrations of amino acids were measured in response to infusion of veratridine (180 μM), a sodium channel activator, as well as during acute noxious stimulation by an injection of 5% formalin into the metatarsal region of the hindleg. Veratridine produced a tetrodotoxin (TTX)‐sensitive increase in the extracellular concentration of Glu. Concentrations of Asp, taurine, Ala, Asn, and Gly were not significantly elevated following veratridine stimulation. In‐tradermal injection of formalin produced a TTX‐sensitive increase in Asp concentration and a non‐TTX‐sensitive increase in Glu concentration. These data support the hypothesis that Glu and Asp are dorsal horn neurotransmitters involved in nociception.
Interaction Between S‐100 Proteins and Steady‐State and Taxol‐Stabilized Microtubules In Vitro
Authors: Donato Rosario , Giambanco Ileana
Published: October 2006

Abstract S‐100 proteins are a group of three 21‐kilodalton, acidic, Ca2+‐binding proteins of the “E‐F hand” type shown to regulate several cell activities, including microtubule (MT) assembly‐disassembly. We show here that S‐100 proteins interact with MTs assembled from either whole microtubule protein or purified tubulin, both in the absence and in the presence of the MT‐stabilizing drug taxol. Evidence for the binding of S‐100 to MTs comes from both kinetic (turbidi‐metric) and binding studies. Kinetically, S‐100 enhances the disassembly of steady‐state MTs in the presence of high concentrations of colchicine or vinblastine at 10 μM free Ca2+ and disassembles taxol‐stabilized MTs at high Ca2+ concentrations. Experiments performed using 125I‐labeled S‐100 show that S‐100 binds Ca2+ independently to a single set of sites on taxol‐stabilized MTs assembled from pure tubulin with an affinity of 6 X 10‐5M and a stoichiometry of 0.15 mol of S‐100/mol of polymerized tubulin. Under certain conditions, S‐100 proteins also cosediment with MTs prepared by coassembly of S‐100 with MTs, probably in the form of an S‐100‐tubulin complex. Because S‐100 binds to MTs under conditions where this protein fraction does not produce observable effects on the kinetics of assembly‐disassembly, e.g., in the absence of Ca2+ at pH 6.7, we conclude that the S‐100 binding to MTs does not affect the stability of MTs per se, but rather creates conditions for increased sensitivity of MTs to Ca2+.
Further Characterization of Phasic Calcium Influx in Rat Cerebrocortical Synaptosomes: Inferences Regarding Calcium Channel Type(s) in Nerve Endings
Authors: Suszkiw J. B. , Murawsky M. M. , Shi M.
Published: October 2006

Abstract Under conditions minimizing the contribution of Na+/Ca2+ exchange to calcium entry in synaptosomes, the K+ depolarization‐dependent calcium influx (JCa) is a single exponential function of time. JCa activates and slowly inactivates at membrane potentials positive to –50 mV, a result indicating the involvement of moderate voltage‐activating, slowly inactivating calcium channels. Calcium channels in synaptosomes are characterized by stronger sensitivity to blockage by Cd2+ than Co2+, insensitivity to dihydropyridine calcium antagonists or the agonist Bay K 8644, and weak, partial sensitivity to the peptide toxin ω‐conotoxin GVIA. These characteristics suggest that voltage‐sensitive calcium channels in rat cerebrocortical synaptosomes are dissimilar from the somatic T, N, or L channel types. JCa is not affected by treatment of synaptosomes with the adenylate cyclase activator forskolin, the membrane permeant dibutyryl‐cyclic AMP, or the kinase C activator phorbol 12‐myristate 13‐acetate diester, results suggesting that calcium channels in synaptosomes are not directly modulated by protein kinase A‐ or C‐mediated phosphorylation.
Purification of Two Dipeptidyl Aminopeptidases II from Rat Brain and Their Action on Proline‐Containing Neuropeptides
Authors: Mentlein Rolf , Struckhoff Gemot
Published: October 2006

Abstract From the soluble and membrane fractions of rat brain homogenate, two enzymes that liberate dipeptides of the type Xaa‐Pro from chromogenic substrates were purified to homogeneity. The two isolated dipeptidyl peptidases had similar molecular and catalytic properties: For the native proteins, molecular weights of 110,000 were estimated; for the denatured proteins, the estimate was 52,500. Whereas the soluble peptidase yielded one band of pi 4.2 after analytical isoelectric focusing, two additional enzymatic active bands were detected between pi 4.2 and 4.3 for the membrane‐associated form. As judged from identical patterns after neuraminidase treatment, both peptidases contained no sialic acid. A pH optimum of 5.5 was estimated for the hydrolysis of Gly‐Pro‐ and Arg‐Pro‐nitroanilide. Substrates with alanine instead of proline in the penultimate position were hydrolyzed at comparable rates. Acidic amino acids in the ultimate N‐terminal position of the substrates reduced the activities of the peptidases 100‐fold as compared with corresponding substrates with unblocked neutral or, especially, basic termini. The action of the dipeptidyl peptidase on several peptides with N‐terminal Xaa‐Pro sequences was investigated. Tri‐peptides were rapidly hydrolyzed, but the activities considerably decreased with increasing chain length of the peptides. Although the tetrapeptide substance P 1‐4 was still a good substrate, the activities detected for the sequential liberation of Xaa‐Pro dipeptides from substance P itself or casomorphin were considerably lower. Longer peptides were not cleaved. The peptidases hydrolyzed Pro‐Pro bonds, e.g., in bradykinin 1‐3 or 1‐5 fragments, but bradykinin itself was resistant. The enzymes were inhibited by serine protease inhibitors, like diisopropyl fluorophosphate or phenylmethylsulfonyl fluoride, and by high salt concentrations but not by the ami‐nopeptidase inhibitors bacitracin and bestatin. Based on the molecular and catalytic properties, both enzymes can be classified as species of dipeptidyl peptidase II (EC 3.4.14.2) rather than IV (EC 3.4.14.5). However, some catalytic properties differentiate the brain enzyme from forms of dipeptidyl peptidase II of other sources.
Structural Properties of the Heparan Sulfate Proteoglycans of Brain
Authors: Ripellino James A. , Margolis Richard U.
Published: October 2006

Abstract: The heparan sulfate proteoglycans present in a deoxycholate extract of rat brain were purified by ion exchange chromatography, affinity chromatography on lipoprotein lipase agarose, and gel filtration. Heparitinase treatment of the heparan sulfate proteoglycan fraction (containing 86% heparan sulfate and 10% chondroitin sulfate) that was eluted from the lipoprotein lipase affinity column with 1 M NaCl led to the appearance of a major protein core with a molecular size of 55,000 daltons, as determined by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. Comparison of the effects of heparinase and heparitinase treatment revealed that the heparan sulfate proteoglycans of brain contain a significant proportion of relatively short N‐sulfoglucosaminyl 6‐O‐sulfate [or N‐sulfoglucosaminyl](α1–4)iduronosyl 2‐0‐sulfate(α 1–4) repeating units and that the portions of the heparan sulfate chains in the vicinity of the carbohydrate‐protein linkage region are characterized by the presence of d‐glucuronic acid rather than l‐iduronic acid. After chondroitinase treatment of a proteoglycan fraction that contained 62% chondroitin sulfate and 21% heparan sulfate (eluted from lipoprotein lipase with 0.4 M NaCl), the charge and density of a portion of the heparan sulfate‐containing proteoglycans decreased significantly. These results indicate that a population of “hybrid” brain proteoglycans exists that contain both chondroitin sulfate and heparan sulfate chains covalently linked to a common protein core.
Compared Effects of Two Vesicular Acetylcholine Uptake Blockers, AH5183 and Cetiedil, on Cholinergic Functions in Torpedo Synaptosomes: Acetylcholine Synthesis, Choline Transport, Vesicular Uptake, and Evoked Acetylcholine Release
Authors: Gaudry‐Talarmain Yvette Morot , Diebler Marie‐Françoise , O'Regan Seana
Published: October 2006

Abstract: We examined the effects of two drugs, AH5183 and cetiedil, demonstrated to be potent inhibitors of acetylcholine (ACh) transport by isolated synaptic vesicles on cholinergic functions in Torpedo synaptosomes. AH5183 exhibited a high specificity toward vesicular ACh transport, whereas cetiedil was shown to inhibit both high‐affinity choline uptake and vesicular ACh transport. Choline acetyltransferase was not affected by either drug. High external choline concentrations permitted us to overcome cetiedil inhibition of high‐affinity choline transport, and thus synthesis of [14C]ACh in treated preparations was similar to that in controls. We then tested evoked ACh release in drug‐treated synaptosomes under conditions where ACh translocation into the vesicles was blocked. We observed that ACh release was impaired only in cetiedil‐treated preparations; synaptosomes treated with AH5183 behaved like the controls. Thus, this comparative study on isolated nerve endings allowed us to dissociate two steps in drug action: upstream, where both AH5183 and cetiedil are efficient blockers of the vesicular ACh translocation, and downstream, where only cetiedil is able to block the ACh release process.
1‐Aminocyclopropane Carboxylic Acid: A Potent and Selective Ligand for the Glycine Modulatory Site of the N‐Methyl‐d‐Aspartate Receptor Complex
Authors: Marvizón Juan‐Carlos G. , Lewin Anita H. , Skolnick Phil
Published: October 2006

Abstract: 1‐Aminocyclopropane carboxylic acid (ACPC) competitively inhibited (IC50, 38 ± 7 nM) [3H]glycine binding to rat forebrain membranes but did not affect [3H]strychnine binding to rat brainstem/ spinal cord membranes. Like glycine, ACPC enhanced 3H‐labelled (+)‐5‐methyl‐10,11 ‐dihydro‐5H‐dibenzo[a, d]cyclohepten‐5,10‐imine maleate ([3H]MK‐801) binding to TV‐methyl‐D‐aspartate receptor‐coupled cation channels (EC50, 135 ± 76 nM and 206 ± 78 nM for ACPC and glycine, respectively) but was ∼ 40% less efficacious in this regard. The maximum increase in [3H]MK‐801 binding produced by a combination of ACPC and glycine was not different from that elicited by glycine, but both compounds potentiated glutamate‐stimulated [3H]MK‐801 binding. These findings indicate that ACPC is a potent and selective ligand at the glycine modulatory site associated with the N‐methyl‐d‐aspartate receptor complex.
Phosphorylation of Superior Cervical Ganglion Proteins During Regeneration
Authors: Watterson J. G. , Good R. , Moses E. , Hearn M. T. W. , Austin L.
Published: October 2006

Abstract: The incorporation of radioactive phosphate into proteins of both normal and regenerating ganglia of the sympathetic nervous system of the rat is reported. The incorporation reactions were carried out in vitro by incubating homogenates of excised ganglia with [γ‐32P]ATP under various conditions. It was found that incorporation of phosphate into proteins of regenerating ganglia in the molecular mass range 10,000–100,000 daltons increased up to 40% over incorporation into proteins from control ganglia during the first 3 days following injury and returned to control levels after 14 days. Analysis of the proteins by two‐dimensional electrophoresis revealed that only few, i.e., <20, became radioactively labelled in homogenates of superior cervical ganglia in the presence of Ca2+, and even fewer in the presence of cyclic AMP. Furthermore, all these proteins fell within a narrow pI range of 4–6. The growth‐associated protein, variously designated GAP‐43, B‐50, F‐1, and pp46, has an enhanced level of expression and phosphorylation in regenerating ganglia compared with controls at day 3. Injury also caused consistently higher levels of incorporation into two other proteins with molecular masses at positions 55,000 and 85,000 and pI values of 5.1 and 4.5, respectively; the former protein most probably is β‐tubulin. The fact that both proteins are found in the 15,000 g pellet after the tissue has been solubilized in 0.5% nonionic detergent indicates that they may indeed be components of filament assemblies. Thus, the results suggest that protein phosphorylation is a mechanism involved in cytoskeletal function in regenerating nerve.
Metabolism of Prostaglandin D2 by Human Cerebral Cortex into 9α, 11β‐Prostaglandin F2 by an Active NADPH‐Dependent 11‐Ketoreductase
Authors: Wolfe L. S. , Rostworowski K. , Pellerin L. , Sherwin A.
Published: October 2006

Abstract: In homogenates of rat cerebral neocortex prostaglandin D2 (PGD2) was found to be quantitatively the main PG biosynthesized by a cytosolic PGD synthetase from en‐dogenously released arachidonic acid. Amounts of 628 ng/g wet weight were found after 30‐min incubation periods compared with basal levels of 2.3 ng/g wet weight. In human cerebral cortex, whether obtained at biopsy or postmortem, only small amounts of PGD2 (4.5–11.7 ng/g wet weight/30 min) were formed. Furthermore, PGD2, added to homogenates of human biopsy temporal cortex, was converted efficiently into 9α,11β‐PGF2 by a NADPH‐dependent 11‐ke‐toreductase as has been reported in other human tissues (liver and lung). PGF2α was determined directly as the fl‐butylbo‐ronate derivative. It became clear that 9α,11β‐PGF2 was formed in considerably greater amounts than PGF2α and that other metabolites are also formed. These results can account for the low amounts of PGD2 found in incubations of human brain tissue. The rat brain does not contain 11‐ketoreductase activity. The present results indicate that the 9α, 11β‐PGF2 must be considered along with other eicosanoids in pathophysiological situations in brain.
Bradykinin Stimulates Phosphoinositide Hydrolysis and Mobilization of Arachidonic Acid in Dorsal Root Ganglion Neurons
Authors: Gammon Charles M. , Allen Ann C. , Morell Pierre
Published: October 2006

Abstract: Cultures of fetal rat dorsal root ganglion neurons (7 days in culture) were prelabeled with myo‐[3H]inositol or [3H]arachidonic acid for 24 h and stimulated with 10 μM bradykinin for time intervals of 5–300 s. The incubation was terminated by addition of 5% perchloric acid to extract inositol phosphates or organic solvent to extract lipids. Inositol phosphates were resolved by anion‐exchange HPLC; lipids were resolved by TLC. Bradykinin stimulation resulted in a 10‐fold increased accumulation of inositol 1,4,5‐trisphosphate (IP3) and inositol bisphosphate (IP2) (fivefold) by 5 s. The increase in IP3 was transient (half maximal by 1 min), whereas stimulated IP2 levels were sustained for several minutes. Even longer term increases were observed in inositol monophosphate. Stimulation also resulted in a threefold increase in arachidonic acid which was preceded by transient increases in diacylglycerol (twofold) and arachidonoyl‐monoacylgly‐cerol (threefold). The temporal lag in the accumulation of arachidonic acid with respect to diglyceride and monoglyc‐eride suggested the involvement of di‐ and monoglyceride lipases in arachidonic acid mobilization. A role for phospho‐lipase A2 is also possible, because pretreatment of cultures with quinacrine partially blocked arachidonic acid release. Bradykinin‐stimulated arachidonic acid release was decreased in the presence of calcium channel blockers nifedipine or verapamil (50 μM), or EDTA (2.5mM). The role of calcium was verified further in that accumulation of phosphatidic acid, diacylglycerol, and arachidonic acid was maximally stimulated by treatment with the calcium ionophore A23187 (20 μM)
Brain and Spinal Cord Levels of Histamine in Lewis Rats with Acute Experimental Autoimmune Encephalomyelitis
Authors: Orr Edward L. , Stanley Nancy C.
Published: October 2006

Abstract: Acute experimental autoimmune encephalomyelitis (EAE) was induced in Lewis rats by inoculation with guinea pig spinal cord homogenate emulsified with Mycobacterium tuberculosis‐enriched complete Freund's adjuvant (CFA). Control rats were inoculated with CFA alone. Control and EAE rats were killed on days 7,9, 11, and 13 postinoculation, and regional brain and spinal cord levels of histamine were determined. No regional differences in histamine content between control and EAE rats were seen on day 7 or 9 post‐inoculation. However, depending on the region, EAE rats exhibited significantly higher levels of histamine in their CNS on day 11 or 13 postinoculation or on both. Thus, regionally and temporally specific increases in brain and spinal cord levels of histamine develop concomitant with or just after the appearance (on day 10 postinoculation) of clinical signs of acute EAE, a finding suggesting that histamine may be involved in the development or expression of acute EAE in Lewis rats.
Regulation of Substance P Receptor Affinity by Guanine Nucleotide‐Binding Proteins
Authors: Macdonald S. G. , Boyd N. D.
Published: October 2006

Abstract: The binding of substance P (SP) to receptors in peripheral tissues as well as in the CNS is subject to regulation by guanine nucleotides. In this report, we provide direct evidence that this effect is mediated by a guanine nucleotide‐binding regulatory protein (G‐protein) that is required for high‐affinity binding of SP to its receptor. Rat submaxillary gland membranes bind a conjugate of SP and I25I‐labeled Bolton‐Hunter reagent (125I‐BHSP) with high affinity (KD= 1.2 ± 0.4 × 10−9M) and sensitivity to guanine nucleotide inhibition. Treatment of the membranes with alkaline buffer (pH 11.5) causes a loss of the high‐affinity, GTP‐sensitive binding of l25I‐BHSP and a parallel loss of [35S]guanosine 5′‐(3‐O‐thio)triphosphate ([35S]GTPγS) binding activity. Addition of purified G‐proteins from bovine brain to the alkaline‐treated membranes restores high‐affinity 125I‐BHSP binding. Reconstitution is maximal when the G‐proteins are incorporated into the alkaline‐treated membranes at a 30‐fold stoichiometric excess of GTPγS binding sites over SP binding sites. Both Go (a pertussis toxin‐sensitive G‐protein having a 39,000‐dalton α‐subunit) and Gi (the G‐protein that mediates inhibition of adenylate cyclase) appear to be equally effective, whereas the isolated α‐subunit of Go is without effect. The effects of added G‐proteins are specifically reversed by guanine nucleotides over the same range of nucleotide concentrations that decreases high‐affinity binding of 125I‐BHSP to native membranes. Although our results indicate that SP receptors in rat submaxillary gland membranes are coupled to a G‐protein that possesses a nucleotide specificity similar to that of Go/Gi, the relevant G‐protein appears to differ from Go and Gi in terms of its sensitivity to pertussis toxin treatment. Reconstitution methods described should be useful in future studies to purify this G‐protein and to analyze further its interaction with the SP receptor.
In Vivo Determination of 5‐Hydroxytryptamine Receptor‐Stimulated Phosphoinositide Turnover in Rat Brain
Authors: Hide Izumi , Kato Tadahiro , Yamawaki Shigeto
Published: October 2006

Abstract: Phosphoinositide turnover stimulated by 5‐hy‐droxytryptamine (5‐HT) receptors in the intact rat brain was studied using an in vivo method. Phosphoinositides in the rat brain were prelabeled with [3H]inositol injected into the lateral cerebral ventricles. The rats were killed by microwave irradiation after 48 h and the contents in the frontal cortex of 3H‐inositol phosphates, [3H]inositol‐1‐monophosphate ([3H]IP1), [3H]inositol‐l,4‐bisphosphate ([3H]IP2), and a mixture of [3H]inositol‐l,4,5‐trisphosphate and [3H]inositol‐1,3,4‐trisphosphate ([3H]IP3) were assayed by HPLC. Lithium treatment (10 mEq/kg, i.p., 2 h before) increased the content of[3H]IP1 and[3H]IP2. 5‐Methoxy‐N′,N‐dimethyltryptamine (5‐MeODMT) and quipazine, 5‐HT agonists, significantly increased the amount of 3H‐inositol phosphates under lithium pretreatment. The response to 5‐MeODMT was inhibited by ritanserin, a 5‐HT2 antagonist, but not by (—)‐propranoloI, a 5‐HT1 antagonist. These results suggest that phosphoinositide turnover in the rat frontal cortex in vivo is stimulated by 5‐HT2 receptor activation. It is considered that this method will be useful for measurement of 5‐HT2 receptor‐stimulated phosphoinositide turnover in vivo to examine the in vivo effects of various psychotropic drugs such as antidepressants.
Capsaicin‐Induced Ion Fluxes Increase Cyclic GMP but Not Cyclic AMP Levels in Rat Sensory Neurones in Culture
Authors: Wood J. N. , Coote P. R. , Minhas A. , Mullaney I. , McNeill M. , Burgess G. M.
Published: October 2006

Abstract: Capsaicin, which induces fluxes of sodilim, calcium, and potassium ions in a subset of both neonatal and adult rat dorsal root ganglion neurones, increased cyclic GMP (cGMP) levels by a factor of 20 (EC50 0.07 μ) to 10‐20 pmol cGMP/mg protein in these cells. Cyclic AMP (CAMP) levels were unaffected. Nonneuronal cells derived from rat ganglia, and both neurones and nonneuronal cells from chick were unresponsive to capsaicin. Capsaicin‐induced cGMP elevation in rat dorsal root ganglion (DRG) neurones was unaffected by pertussis toxin, lowered by compounds that block voltage‐sensitive calcium channels, and was abolished by the removal of extracellular calcium. Calcium, guanidine, and rubidium fluxes were unaffected by treatment of DRG cells with sodium nitroprusside or dibutyryl cGMP. The cGMP response to capsaicin is thus a function of capsaicin‐evoked calcium uptake through voltage‐sensitive calcium channels. Elevated cGMP levels do not, however, contribute to capsaicin‐evoked ion fluxes or to their desensitisation.
Heterogeneity of Soluble Neural Cell Adhesion Molecule
Authors: Nybroe Ole , Linnemann Dorte , Bock Elisabeth
Published: October 2006

Abstract: Soluble neural cell adhesion molecule (NCAM) from rat brain neuronal cell culture media consists predominantly of a polypeptide of Mr∼ 115,000. Minor amounts of a polypeptide of Mr∼ 180,000 and two inconsistently appearing components of Mr 160,000 and 145,000 are also observed. The Mr 115,000 component is derived from the neuronal membrane NCAM components NCAM‐A of Mr 190,000, NCAM‐B of Mr 140,000, or both. Thus, as a part of the catabolism of membrane NCAM‐A plus ‐B, a minor fraction is posttranslationally cleaved and recovered in the media as discernible soluble NCAM polypeptides. The half‐life of membrane NCAM‐A plus ‐B is <24 h. Astrocyte culture media contains a predominant soluble NCAM component of Mr 120,000 derived from membrane‐associated NCAM‐C. A close comparison of deglycosylated soluble NCAM from astrocyte and neuronal cultures showed a small but consistent difference in Mr, a result suggesting that different NCAM polypeptides are released from the membrane of neurons and astrocytes. In contrast to the Mr 115,000‐120,000 NCAM polypeptides, the Mr 180,000 polypeptide from neuronal culture media does not seem to be derived from membrane‐attached NCAM and may therefore represent a secreted NCAM isoform
Effects of a New Calcium Channel Blocker, KB‐2796, on Protein Synthesis of the CA1 Pyramidal Cell and Delayed Neuronal Death Following Transient Forebrain Ischemia
Authors: Yoshidomi Mikio , Hayashi Takashi , Abe Koji , Kogure Kyuya
Published: October 2006

Abstract: The effects of a new calcium channel blocker, l‐[bis(4‐fluorophenyl)methyl]‐4‐(2,3,4‐trimethoxybenzyl)‐piperazine dihydrochloride (KB‐2796), on delayed neuronal death (DND) in the hippocampus were examined in gerbils in comparison with those of pentobarbital and flunarizine. The neuronal density in the hippocampal CA1 subfield was counted on the seventh day of recirculation following 5 min of bilateral carotid occlusion, and protein biosynthesis in the brain was also determined at 1,2, 4, 24, and 72 h following occlusion. The drugs were intraperitoneally administered after recirculation. KB‐2796 (10 mg/kg) significantly prevented DND in the CA1 subfield. Pentobarbital (40 mg/kg), but not flunarizine (3 and 10 mg/kg), inhibited DND. Protein synthetic activity in the CA1 subfield was reduced by ischemia and the reduction was not restored even at 72 h after recir culation. KB‐2796 did not ameliorate the reduction of protein synthesis in the CA1 subfield by 24 h after recirculation, but in one of three animals restoration of protein synthesis was observed at 72 h of recirculation. Pentobarbital also restored the reduced protein synthesis in two of three animals at 72 h. These results suggest that calcium influx into neurons participates in the pathogenesis of DND, and also that KB‐2796 might prevent both morphological and functional cell damage in CA1 neurons induced by transient ischemia.
Stimulation by ATP of Inositol Trisphosphate Accumulation and Calcium Mobilization in Cultured Adrenal Chromaffin Cells
Authors: Sasakawa Nobuyuki , Nakaki Toshio , Yamamoto Satoshi , Kato Ryuichi
Published: October 2006

Abstract: Effects of ATP on accumulation of inositol phosphates and Ca2+ mobilization were investigated in cultured bovine adrenal chromaffin cells. When the cells were stimulated with 30 μM ATP, a rapid and transient rise in intracellular Ca2+ concentration was observed. At the same time, ATP rapidly increased accumulation of inositol phosphates. The concentration–response curve for the ATP‐induced Ca2+ mobilization was similar to that for inositol trisphosphate (IP3) accumulation. ATP exerted its maximal effects at 30 μM for either IP3 accumulation or Ca2+ mobilization. The order of the efficacy of the agonists for IP3 accumulation and Ca2+ mobilization at 100 μM was ATP > ADP > AMP ± adenosine. AMP (100 μM) and adenosine (300 μM) failed to induce IP3 accumulation and Ca2+ mobilization. Although 100 μM GTP and 100 μM UTP also induced IP3 accumulation and Ca2+ mobilization, their efficacy was less than that of ATP. CTP (100 μM) induced a slight IP3 accumulation, but it did not induce Ca2+ mobilization. Nifedipine (10 μM), a Ca2+ channel antagonist, and theophylline (100 μM), a P1‐purinergic receptor antagonist, failed to inhibit the ATP‐induced IP3 accumulation and Ca2+ mobilization. The above two cellular responses induced by ATP were also observed in the Ca2+‐depleted medium. ATP induced a rapid and transient accumulation of 1,4,5‐IP3 (5 s), followed by a slower accumulation of 1,3,4‐IP3. These results suggest that ATP induces the formation of 1,4,5‐IP3 through the P2‐purinergic receptor and consequently promotes Ca2+ mobilization from intracellular storage sites in cultured adrenal chromaffin cells.
Ganglioside Composition of Normal and Mutant Mouse Embryos
Authors: Bouvier Joseph D. , Seyfried Thomas N.
Published: October 2006

Abstract: The enrichment of gangliosides in neuronal membranes suggests that they play an important role in CNS development. We recently found a marked tetrasialoganglioside deficiency in tw1/tw1 mutant mouse embryos at embryonic day (E)‐11. The recessive tw1/tw1 mutants die at embryonic ages E‐9 to E‐18 from failed neural differentiation in the ventral portion of the neural tube. In the present study, we examined the composition and distribution of gangliosides in tw1/tw1 mutant mouse embryos at E‐12. The total ganglioside sialic acid concentration was significantly lower in the mutants than in normal (+/‐) embryos. The mutants also expressed significant deficiencies of gangliosides in the “b′’ metabolic pathway (GD3, GD1b, GT1b, and GQ1b) and elevations in levels of gangliosides in the “a′’ metabolic pathway (GM3, GM2, GM1, and GD1a). These findings suggest that the mutants have a partial deficiency in the activity of a specific sialyltransferase in the b pathway. Regional ganglioside distribution was also studied in E‐12 normal mouse embryos. The ganglioside composition in heads and bodies was similar to each other and to whole embryos. Total ganglioside concentration and the distribution of b pathway gangliosides were significantly higher in neural tube regions than in nonneural tube regions. These findings suggest that b pathway gangliosides accumulate in differentiating neural cells and that the deficiency of these gangliosides in the tw1/tw1 mutants is closely associated with failed neural differentiation.
Hippocampal and Cerebellar β‐Adrenergic Receptors and Adenylate Cyclase Are Differentially Altered by Chronic Ethanol Ingestion
Authors: Valverius Peter , Hoffman Paula L. , Tabakoff Boris
Published: October 2006

Abstract: Chronic ethanol ingestion by mice resulted in the loss of high‐affinity β‐adrenergic agonist binding sites and a significant decrease in activation of adenylate cyclase by guanine nucleotides and β‐adrenergic agonists in the hippocampus, although no significant change was noted in the total number of β‐adrenergic receptors, as defined by the binding of the antagonist [125I]iodocyanopindolol. In cerebellum, chronic ethanol ingestion resulted in a 16% decrease in the total concentration of β‐adrenergic receptors and in a decrease in the affinity for agonist of the high‐affinity β‐adrenergic agonist binding sites. However, neither the amount of the high‐affinity agonist binding sites nor the activation of adenylate cyclase by agonist was affected. The different responses to ethanol in hippocampus and cerebellum may result from quantitative differences in distribution of β1‐ and β2‐adrenergic receptors in the tested brain areas and/or differential effects of ethanol on stimulatory guanine nucleotide binding protein in these brain areas.
Inactivation of Cyclic AMP‐Dependent Taurine Release from Astroglia
Authors: Shain William , Madelian Vergine , Martin David L.
Published: October 2006

Abstract: When astroglial cells are exposed to β‐adrenergic agonists for long periods of time (>20 min), transient increases in taurine release and intracellular cyclic AMP (cAMP) are observed. Three phases of taurine release can be distinguished: activation, inactivation, and an elevated steady state. In this article, we present data describing the relationship between intracellular cAMP levels and inactivation of taurine release. To do this, we compared the apparent first‐order rate constants for the inactivation of taurine release (ktau) with the apparent first‐order rate constant for the decline of intracellular cAMP (kCAMP). We also measured ktau under experimental conditions that were chosen to provide a wide range of intracellular cAMP concentrations or to stimulate release without the involvement of the β‐adrenergic receptor and adenylate cyclase. When taurine release was stimulated with a saturating concentration of isoproterenol, the inactivation of release was significantly faster than the decline of intracellular cAMP. Furthermore, there were no significant differences in ktau measured under any of the experimental conditions used. Thus, inactivation of taurine release does not involve changes in the activity of the β‐adrenergic receptor and adenylate cyclase, i.e., desensitization, and appears to be independent of the intracellular concentration of cAMP. These results indicate that cAMP‐mediated events can be regulated by mechanism(s) in addition to those that control receptor‐adenylate cyclase interactions and the synthesis of cAMP. Such regulation may occur by inactivation of protein kinase A activity or the taurine transport mechanism or by activation of a phosphoprotein phosphatase.
Inositol Lipid Metabolism in Rat Hippocampal Formation Slices: Basal Metabolism and Effects of Cholinergic Agonists
Authors: Stephens L. R. , Logan S. D.
Published: October 2006

Abstract: Incubation of rat hippocampal formation slices under steady‐state conditions with [3H]inositol leads to only three phospholipids becoming labelled: phosphatidylinositol, phosphatidylinositol 4‐phosphate, and phosphatidylinositol 4.5‐bisphosphate. All three lipids incorporate [32P]Pi into their phosphodiester phosphate group with the polyphosphoinositides also incorporating this tracer into their monoester phosphate groups. As the concentrations of these lipids remain constant during these labelling processes we conclude that the phosphodiester phosphate, the inositol moiety, and the monoester phosphate groups undergo metabolic turnover in hippocampal formation slices incubated in vitro. The rate of incorporation of [3H]inositol into all three inositol phospholipids was stimulated by the addition of methacholine to the medium. Moreover, following steady‐state labelling of the inositol lipids with [3H]inositol, methacholine in the presence of 10 mM LiCl caused a transient fall of 13% in the radiochemical concentration of phosphatidylinositol 4,5‐bisphosphate after only 30 s stimulation and a fall of 15% in the radiochemical concentration of phosphatidylinositol after 30 min. Concomitantly, there was an approximately stoichiometric rise in the radiochemical concentration of inositol phosphates. Thus, we suggest that methacholine stimulates an inositol phospholipid phosphoinositidase C in rat hippocampal formation slices.
Dendrotoxin, 4‐Aminopyridine, and β‐Bungarotoxin Act at Common Loci but by Two Distinct Mechanisms to Induce Ca2+‐Dependent Release of Glutamate from Guinea‐Pig Cerebrocortical Synaptosomes
Authors: Tibbs Gareth R. , Dolly J. Oliver , Nicholls David G.
Published: October 2006

Abstract: The release of endogenous glutamate from guineapig cerebrocortical synaptosomes evoked by dendrotoxin, β‐bungarotoxin, and 4‐aminopyridine is compared. Dendrotoxin and 4‐aminopyridine cause Ca2+‐dependent release, representing a partial depletion of the KCl‐releasable transmitter pool. The decrease in the plasma membrane potential caused by 4‐aminopyridine or dendrotoxin and the evoked release of glutamate from a transmitter pool accord with the inhibitory action of these agents on certain K+ conductances. In contrast, the massive release of glutamate evoked by β‐bungarotoxin is produced in the presence of Ca2+ but not of Sr2+, a result consistent with a generalised permeabilisation of synaptosomal plasma membranes. Although dendrotoxin inhibits the binding of β‐bungarotoxin and the resultant synaptosomal lysis, demonstration of a direct effect of β‐bungarotoxin binding per se on K+ permeability is impractical owing to its phospholipase A2 activity.
A Selective Increase in Particulate Superoxide Dismutase Activity in Parkinsonian Substantia Nigra
Authors: Saggu H. , Cooksey J. , Dexter D. , Wells F. R. , Lees A. , Jenner P. , Marsden C. D.
Published: October 2006

The total activity of superoxide dismutase (SOD) and cytosolic and particulate activity of SOD in human substantia nigra and cerebellum were measured by a spectrophotometric method based on the ability of SOD to inhibit the autoxidation of adrenaline. The cystosolic and particulate isoenzymes of SOD were differentiated by the inclusion of potassium cyanide which selectively inhibits cytosolic copper/zinc‐dependent SOD activity. In autopsied human brains, there was no difference in total SOD) activity, or the activity of SOD in cytosol in substantia nigra jof patients dying with Parkinson's disease compared to age‐matched controls. However, the activity of the particulate form of SOD was higher in the parkinsonian substantia nigra compared to control tissue. In the cerebellum there was no difference in the total, cytosolic, or particulate activity of SOD between parkinsonian patients and age‐matched controls. Increased activity of SOD in particulate fraction may be a protective response to elevated levels of toxic free radicals in the parkinsonian substantia nigra. Alternatively, increased SOD activity may induce cell death through the accumulation of hydrogen peroxide.
Glycine Potentiates the Stimulation of Inositol Phospholipid Hydrolysis by Excitatory Amino Acids in Primary Cultures of Cerebellar Neurons
Authors: Nicoletti Ferdinando , Canonico Pier Luigi
Published: October 2006

Glycine potentiates stimulation of inositol phospholipid hydrolysis by glutamate and N‐methyl‐D‐aspartate, but not by quisqualate or carbamylcholine, in primary cultures of cerebellar granule cells. This potentiation occurs in the absence of extracellular Mg2+, but is more evident when stimulation of inositol phospholipid hydrolysis by N‐methyl‐D‐aspartate is measured in the presence of 1 mM Mg2+. The action of glycine is not antagonized by strychnine. These results suggest that glycine acts as a positive modulator of signal transduction at a specific class of N‐methyl‐D‐aspartate‐sensitive glutamate receptors coupled to inositol phospholipid hydrolysis in cerebellar granule cells.
Phosphorylation of P400 Protein by Cyclic AMP‐Dependent Protein Kinase and Ca2+/Calmodulin‐Dependent Protein Kinase II
Authors: Yamamoto Hideyuki , Maeda Nobuaki , Niinobe Michio , Miyamoto Eishichi , Mikoshiba Katsuhiko
Published: October 2006

Purified P400 protein was phosphorylated by both purified Ca2+/calmodulin‐dependent protein kinase II (CaM kinase II) and the catalytic subunit of cyclic AMP‐dependent protein kinase (A‐kinase). Because P400 protein was suggested to function as an integral membrane protein, we investigated the phosphorylation of P400 protein using crude mitochondrial and microsomal fractions (P2/P3 fraction). Incubation of the P2/P3 fraction from mouse cerebellum with cyclic AMP or the catalytic subunit of A‐kinase stimulated the phosphorylation of P400 protein. The phosphorylation of P400 protein was not observed in the P2/P3 fraction from mouse forebrain. Cyclic AMP and A‐kinase enhanced the phosphorylation of several proteins, including P400 protein, suggesting that P400 protein is one of the best substrates for A‐kinase in the P2/ P3 fraction. Although endogenous and exogenous CaM kinase II stimulated the phosphorylation of some proteins in the P2/P3 fraction, the phosphorylation of P400 protein was weak. Immunoprecipitation with the monoclonal antibody to P400 protein confirmed that the P400 protein itself was definitely phosphorylated by the catalytic subunit of A‐kinase and CaM kinase II. A‐kinase phosphorylated only the seryl residue in P400 protein. Immunoblot analysis of the cells in primary culture of mouse cerebellum confirmed the expression of P400 protein, which migrated at the same position on sodium do‐decyl sulfate‐polyacrylamide gel electrophoresis as that in the P2/P3 fraction. Incubation of the cultured cerebellar cells with [32P]orthophosphate resulted in the labeling of P400 protein. These results suggest that P400 protein is one of the best substrates for A‐kinase and CaM kinase II in cerebellum and that the function of P400 protein is regulated by cyclic AMP‐and Ca2+/calmodulin‐dependent phosphorylation.
Monoclonal Antibody to Embryonic CNS Antigen A2B5 Provides Evidence for the Involvement of Membrane Components at Sites of Alzheimer Degeneration and Detects Sulfatides as Well as Gangliosides
Authors: Majocha Ronald E. , Jungalwala Firoze B. , Rodenrys Anne , Marotta Charles A.
Published: October 2006

Immunohistological and biochemical studies were initiated to determine whether or not neural membrane components were associated with degenerative changes characteristic of Alzheimer's disease (AD). Monoclonal antibody A2B5, developed against embryonic chick retinal cells and previously shown to react with neural surface gangliosides, was applied to formalin‐fixed sections of control and AD brain tissue. Frontal cortex and hippocampus of AD cases exhibited high levels of A2B5 immunoreactivity within those neurons undergoing neurofibrillary degeneration. Neuritic processes associated with senile plaques were also highly reactive with the A2B5 antibody. The amount of gangliosides and their pattern after HPTLC were the same in control and AD cases. However, the unexpected observation was made that the A2B5 antibody reacted with human brain sulfatides in addition to the expected reactivity with minor gangliosides. The average level of sulfatides in AD brain was significantly higher than in normal controls. The data support the involvement of one or more membrane components with neu‐rodegeneration in the Alzheimer brain.
Pharmacological Characteristics and Distributions of σ and Phencyclidine Receptors in the Animal Kingdom
Authors: Vu Tuan H. , Weissman Arthur D. , London Edythe D.
Published: October 2006

The phylogenetic distributions ofσ‐ and phencyclidine receptors in neural tissues of 13 species and the pharmacological characteristics of these receptors in whole sea anemone and neural tissues of the guinea pig, chicken, and frog were studied. Specific binding of [3H]haloperidol and [3H]N‐[1‐(2‐thienyl)cyclohexyl]‐3,4‐piperidine, ligands that bind with high affinity to σ‐ and phencyclidine receptors, respectively, was detected in all organisms examined. The order of potencies of various ligands to inhibit 1 nM [3H]haloperidol binding in brains of frogs and guinea pigs or 1 nM [3H]N‐[1‐(2‐thienyl)cyclohexyl]‐3,4–piperidine in chicken or guinea pig brain homogenates was very similar. However, the characteristics and stereospecificity of binding of the two radioligands in sea anemone were different than in higher organisms. The results suggest that σ– and phencyclidine binding sites are evolutionarily old, as the characteristics of the two sites are well preserved over a range of vertebrate phyla.
Marked Regional Heterogeniety of 125I‐Bolton Hunter Substance P Binding and Substance P‐Induced Activation of Phospholipase C in Astrocyte Cultures from the Embryonic or Newborn Rat
Authors: Beaujouan J. C. , de Montety M. C. Daguet , Torrens Y. , Saffroy M. , Dietl M. , Glowinski J.
Published: October 2006

The specific binding of 125‐Bolton Hunter substance P (125I‐BHSP) was estimated on 4‐ to 5‐week‐old primary cultures of astrocytes from several brain structures and the spinal cord of 16‐day‐old embryonic or newborn rats. In both cases, high levels of binding of 125I‐BHSP were found on intact astrocytes from the brainstem, but this binding was low or negligible on cells from the cerebral cortex, striatum, hypothalamus, and mesencephalon. In addition, hippocampal astrocytes from newborn rats were also devoid of 125I‐BHSP binding sites, while a binding of 125I‐BHSP (half that of brainstem cells) was observed on astrocytes from the cerebellum and spinal cord. It was also shown that this regional heterogeneity in 125I‐BHSP binding was not linked to differences in the inactivation of the ligand, cell plating density. or eventual cell contaminants. Five‐day‐old cultures from 16‐day‐old embryos were used to estimate 125I‐BHSP binding on neuron‐enriched cultures. Specific 125I‐BHSP binding was found on cells from the brainstem, mesencephalon, and hypothalamus, but neurons from the cerebral cortex or the striatum contained low or negligible amounts of 125I‐BHSP binding sites. Competition studies using tachykinins and SP analogues indicated that 125I‐BHSP binding sites on brainstem astrocytes (16‐day‐old embryos) have the pharmacological profile expected for NK1 binding sites. SP (1 μM) stimulated phosphoinositide breakdown in cells rich in 125I‐BHSP binding sites (brainstem) but not in those devoid of 125I‐BHSP binding (striatum). These results are discussed in light of previous observations that have shown the presence of 125I‐BHSP binding sites on astrocytes from different brain structures of the mouse.
Modulation of γ‐Aminobutyric Acid Release in Cerebral Cortex by Fluoride, Phorbol Ester, and Phosphodiesterase Inhibitors: Differential Sensitivity of Acetylcholine Release to Fluoride and K+ Channel Blockers
Authors: Gardiner I. M. , de Belleroche J.
Published: October 2006

In this study we have used fluoride as a tool to investigate the involvement of G protein‐coupled effector systems in the regulation of the depolarization‐induced release of γ‐aminobutyric acid (GABA) from rat cerebral cortex. To distinguish among the activating effects of NaF on G proteins linked to different effectors, such as adenylate cyclase, polyphosphoinositide phospholipase C, and K+ channels, agents specific to these effectors have been used in parallel. NaF induced a marked dose‐dependent facilitation of the K+‐evoked release of [14C]GABA, with an EC50 of 1.26 mM, increasing release by 103% at 5 mM NaF. No effect on basal release was seen up to 3 mM NaF, and no modulation of [3H]acetylcholine (ACh) release was seen up to 5 mM NaF. Phorbol 12,13‐diacetate (PDA) produced a similar dose‐dependent facilitation of the K+‐evoked release of [14C]GABA, potentiating the release of [14C]GABA by 50% at 10 μM PDA. The phosphodiesterase inhibitors, 3‐isobutyl‐1‐methylxanthine (IBMX) and theophylline, inhibited the K+‐evoked release of [14C]GABA, and IBMX reversed the NaF facilitation of GABA release in a dose‐dependent manner (pA2 2.57). The K+ channel blocker (IA current) tetrahydroaminoacridine (THA), which markedly inhibits the K+‐evoked release of [14C]GABA, also reversed the NaF facilitatory effect, but the release of [3H]ACh was less sensitive to the inhibitory effect of THA. On the other hand, the K+ channel blocker, tetraethylammonium, which has no effect on the release of [14C]GABA, caused a significant facilitation of K+‐evoked release of [3H]ACh. From these studies, it is concluded that GABA release in cerebral cortex is subject to regulation by G protein‐linked effector systems that are distinct from those affecting the release of [3H]ACh in cerebral cortex.