bluuebunny/crossref_metadata_2025 huggingface dataset:
Dopamine Synthesis in Synaptosomes: Relation of Autoreceptor Functioning to pH, Membrane Depolarization, and Intrasynaptosomal Dopamine Content
Authors: Saller Charles F. , Salama Andre I.
Published: December 2006

Abstract: Factors affecting dopamine (DA) synthesis in rat striatal synaptosomes were examined by measuring the conversion of [3H]tyrosine (Tyr) to [3H]DA. Any [3H]DA that was synthesized was extracted into a toluene‐based scintillation cocktail and quantitated by liquid scintillation spectrometry. The extraction was facilitated using di‐(2‐ethylhexyl) phosphoric acid (DEHP), a liquid cation exchanger. DA, apomorphine, and other DA agonists were much less potent inhibitors of DA synthesis in striatal synaptosomes at pH 6.2 than at pH 7.2. 3‐(3‐Hydroxyphenyl)‐N‐n‐propylpiperidine (3‐PPP), a putative DA autoreceptor agonist, was inactive at pH 6.2. However, at pH 7.2, 3‐PPP did inhibit DA synthesis. This inhibition was reversed by sulpiride, a DA receptor antagonist, but not by benztropine, a DA uptake blocker, suggesting that 3‐PPP inhibits DA synthesis by stimulating the DA autoreceptor. DA release from synaptosomes was much greater at pH 6.2 than at pH 7.2, most probably because the synaptosomal membrane appears to be depolarized at pH 6.2, as measured by the accumulation of [3H]tetraphenylphosphonium ions. Since tyrosine hydroxylase is inhibited by DA, this finding suggested that low assay buffer pH (i.e., pH 6.2) might interfere with the ability of 3‐PPP and other DA agonists to inhibit DA synthesis, by promoting DA release. Likewise, reserpine and tetrabenazine, compounds which disrupt vesicular DA storage, were much less effective inhibitors of DA synthesis at pH 6.2 (high basal DA release). Moreover, d‐amphetamine and high buffer potassium concentrations, treatments which promote DA release, also interfered with the ability of 3‐PPP to inhibit DA synthesis. Thus, modulation of the release of DA in equilibrium with tyrosine hydroxylase may be a mechanism by which the DA autoreceptor regulates DA synthesis.
Angiotensin II Synthesis Studies in Dissociated Brain Cell Cultures
Authors: Weyhenmeyer James A. , Meyer Joan M. , Watkins James E.
Published: December 2006

Abstract: The biosynthesis of angiotensin II‐like peptide was measured in primary cultured brain cells from the fetal rat. Cells from the whole brains of 20‐day gestational age Sprague‐Dawley rats were dissociated by mild trypsinization and grown for 5 days in supplemented (serum‐free) medium prior to experimental analysis. The time‐dependent incorporation of [3H]proline into newly synthesized angiotensin II‐like peptide was measured by radioimmunoassay using specific angiotensin II antisera. Analysis of radioimmunoassay data revealed an increase in the amount of tritium‐labeled angiotensin II in the crude extract of the brain cells during the first 24 h in culture. The chromatographic character of the angiotensin II‐like peptide was further identified by high pressure liquid chromatography. Elution profiles for newly synthesized angiotensin II were identical to those profiles generated for [3H]angiotensin II and nonradiolabeled angiotensin II standards. To determine the bioactivity of the angiotensin II‐like peptide, fractions of the column‐purified peptide were injected into the region of the rat lateral ventricle via an indwelling cannula. Systolic pressure increased up to 20 mm Hg depending upon the amount of peptide injected. These data clearly support the existence of an endogenous renin‐angiotensin system in dissociated brain cell cultures from the fetal rat, and provide an experimental model for further analysis of the regulatory mechanisms of angiotensin II synthesis.
Characterization of Cyclic AMP‐Dependent Phosphorylation of Neuronal Membrane Proteins
Authors: Rauch Nancy , Roskoski Robert
Published: December 2006

Abstract: We examined the patterns of cyclic AMP‐dependent protein phosphorylation in membranes prepared from rat cortical synaptosomes following gel electrophoresis and autoradiography. We determined the optimum pH (6.2), time (20 s), Mg2+ concentration (10 mM) and cyclic AMP concentration (5 μM) for the reaction. We also found that the detergents Triton X‐100 and gramicidin S enhanced cyclic AMP‐dependent protein phosphorylation. Inhibitors of the Na+, K+ ATPase (ouabain, NaF, vanadate) enhanced protein phosphorylation. This effect occurred in the presence but not in the absence of detergent. The addition of purified bovine brain cyclic AMP‐dependent protein kinase catalytic subunit enhanced membrane protein phosphorylation. The addition of homogeneous neural (bovine brain) and non‐neural (bovine skeletal muscle) cyclic AMP‐dependent protein kinase type II regulatory subunit partially inhibited protein phosphorylation. Both neural and non‐neural regulatory subunits behaved similarly. In addition to cyclic AMP‐dependent phosphorylation, the alpha‐subunit of pyruvate dehydrogenase (Mr= 41,000) is phosphorylated in a cyclic AMP‐independent fashion. We also examined the phosphorylation pattern of membranes prepared from rat heart and found that the number of acceptor substrates was much less than that from the nervous system.
Further Characteristics of the ATP‐Stimulated Uptake of Calcium into Chromaffin Granules
Authors: Burger A. , Niedermaier W. , Langer R. , Bode U.
Published: December 2006

Abstract: The ATP‐stimulated uptake of 45Ca2+ [and [3H](‐)‐noradrenaline ([3H]NA)] into chromaffin granules and that into mitochondria are driven by a protonic gradient ΔμH+, composed of the components ΔpH (concentration gradient of protons) and ΔΨ(electrical potential difference). The granular ATPase pumps protons into the matrix (ΔpH inside acid, ΔΨ positive), but the mitochondrial ATPase ejects protons from the matrix (ΔpH alkaline, ΔΨ negative inside). To show different driving forces of uptake, the rate of the ATP‐stimulated uptake of 45Ca2+ (and [3H]NA) into chromaffin granules was compared with the rate of the ATP‐stimulated uptake of 45Ca2+ into mitochondria (adrenomedullary or rat liver). In the presence of nitrate, the rate of the ATP‐stimulated uptake of 45Ca2+ into chromaffin granules is higher than in the presence of acetate, because the lyotropic anion nitrate stimulates the granular ATPase and increases ΔpH (acid inside). Compared with nitrate, the rate of the ATP‐stimulated uptake of 45Ca2+ into mitochondria is higher in the presence of the proton‐carrying anion acetate, which, after permeation, provides protons for ejection by the ATPase. In the absence of ATP, a valinomycin‐mediated potassium influx (ΔΨ inside positive) stimulates the granular uptake of [3H]NA, which has an electrogenic component, but not the granular uptake of 45Ca2+, which is electroneutral. The electrogenic uptake of 45Ca2+ into mitochondria is stimulated by a valinomycin‐mediated potassium efflux (ΔΨ negative inside). The ATP‐stimulated uptake of 45Ca2+ into chromaffin granules is sensitive to ruthenium red, suggesting a carrier‐mediated mechanism of uptake, and it is sensitive to atractyloside, indicating the simultaneous uptake of ATP. After collapse of ΔpH by ammonia, the ATP‐stimulated uptake of 45Ca2+ into chromaffin granules is abolished, but not that into mitochondria. In the presence of ammonia, the rate of the ATP‐stimulated uptake of [3H]NA is very low, and an ATP‐independent uptake of 45Ca2+ into chromaffin granules is observed which is similar to the ATP‐independent Ca2+/Na+ exchange at the granular membrane.
Alanine Uptake and Release by Sympathetic Ganglia of Chicken Embryos
Authors: Larrabee Martin G.
Published: December 2006

Abstract: Uptake and release of alanine were measured in lumbar sympathetic chains excised from embryos of white leghorn chickens, 14‐15 days old, and incubated in a modified Eagle's minimum essential medium. In the presence of [U‐14C]glucose, glucose carbon accumulated in alanine in the medium at a rate that increased when unlabeled alanine was added and sometimes exceeded the rate of appearance in lactate. When combined with uptake data, the increase in appearance of labeled alanine in the medium could be accounted for quantitatively by interference with its reuptake, without assuming a change in the unidirectional output of labeled alanine, provided allowance was made for the measured properties of exchange between the extracellular space and the surrounding medium. According to this model, the constant unidirectional outflux of labeled alanine was about 50 μmol/g dry weight/h. When [U‐14C]alanine was added to medium containing unlabeled glucose, the alanine was consumed at a rate that increased as the concentration of alanine in the medium was elevated. The uptake rate was found to fit a modified Michaelis‐Menten equation with a Umax of about 120 μmol/g dry weight/h, a Km of 0.5‐1.0 mM, and a Kd of 0.75 ml/g dry weight/h. By chemical measurement of changes in alanine concentration in the medium during incubation, the uptake rate was shown to equal the output rate when about 0.2 mM alanine was present. Much of the alanine consumed in the presence of glucose was metabolized to CO2, raising the total CO2 output above the rate obtained with glucose alone. When alanine was present at a concentration of 10‐20 mM, it contributed almost as much carbon to CO2 as did the glucose. A higher percentage of the carbon from alanine was incorporated into tissue constituents than was carbon from either glucose or lactate. It is concluded that alanine can be significant both as a product and as a substrate, but that its role as substrate would not be great at typical concentrations of alanine in blood.
Determination of 1‐Methylimidazole‐4‐Acetic Acid in Brain Tissue from Rat and Mouse
Authors: Swahn Carl‐Gunnar
Published: December 2006

Abstract: The concentration of the histamine metabolite 1‐methylimidazole‐4‐acetic acid was determined in brain tissue from rat and mouse with a gas chromatographic‐mass spectrometric method. Mouse brain contained 1.7‐3.2 nmol/g, depending on the strain. The concentration in cerebrum from Sprague‐Dawley rats was 1.2 nmol/g, whereas cerebellum contained 0.24 nmol/g. The concentration of tele‐methylhistamine in mouse brain was 1.4‐2.2 nmol/g. The concentration of 1‐methylimidazole‐4‐acetic acid in rat brain after death did not change significantly during 2 h at room temperature.
Persistence of Immunoreactive Neurofilament Protein Breakdown Products in Transected Rat Sciatic Nerve
Authors: Schlaepfer W. W. , Lee C. , Trojanowski J. Q. , Lee V. M.‐Y.
Published: December 2006

Abstract: Alterations occurring in nerve proteins of transected nerves were studied in rat sciatic nerves using polyclonal and monoclonal antibodies to identify and monitor neurofilament (NF) epitopes among nerve proteins following their electrophoresis and transfer to nitrocellulose paper. Immunoblot methods identified NF epitopes in NF triplet proteins (Mr 200,000, 150,000, and 68,000) and in NF nontriplet proteins (all other immunobands below Mr 200,000 and above Mr 40,000). NF triplet and nontriplet proteins were Triton‐insoluble in both untransected and transected nerves. Extensive loss of NF triplet and most nontriplet proteins occurred during the 24‐48‐h period following nerve transection and was attributed to proteolytic degradation. Loss of protease‐labile NF proteins led to a markedly reduced level of NF immunoreactivity in 2‐day transected nerve. NF proteins which survived the 2‐day posttransectional period were considered to represent protease‐stable NF fragments. These fragments persisted in transected nerve for periods of at least 35 days. Most protease‐stable NF fragments which retained immunoreactivity had Mr of 57,000‐65,000. Low concentrations of the same immunobands were present in untransected nerves.
Antiepileptic Agents Affect Hypothalamic β‐Endorphin Concentrations
Authors: Martini Alberto , Sacerdote Paola , Mantegazza Paolo , Panerai Alberto E.
Published: December 2006

Abstract: β‐Endorphin, Met‐enkephalin, substance P, and somatostatin concentrations were evaluated in the hypothalami of rats treated either acutely or chronically (15 days) with sodium valproate, diphenylhydantoin, phenobarbital, or ethosuximide. All of these drugs, with the exception of ethosuximide, induced significant decreases in β‐endorphin concentrations after acute treatment, while only sodium valproate induced a decrease after chronic treatment. The acute and chronic effects of sodium valproate were also produced by aminooxyacetic acid, an inhibitor of γ‐aminobutyric acid (GABA) transaminase, while another GABA transaminase inhibitor, ethanolamine‐O‐sulphate, and THIP, a GABA receptor agonist, were effective after acute administration. Metenkephalin, substance P, and somatostatin concentrations were never affected by the drugs used. The present results, indicating that antiepileptic agents specifically decrease β‐endorphin concentrations, seem to correlate well with the capacity of these agents to blunt the epileptic activity of the peptides tested. Moreover, our data suggest that GABA may be involved in the anticonvulsant‐induced reduction of β‐endorphin concentrations.
Enkephalinergic Markers in Substantia Nigra and Caudate Nucleus from Parkinsonian Subjects
Authors: Llorens‐Cortes C. , Javoy‐Agid F. , Agid Y. , Taquet H. , Schwartz J. C.
Published: December 2006

Abstract: Marked reductions in opiate receptor binding (−42%), “enkephalinase” activity (−39%), and Met5‐enkephalin levels (−72%) accompanied the well‐established dopamine depletion in the substantia nigra pars compacta of Parkinsonian subjects. In contrast, enkephalinergic markers were not significantly modified in caudate nucleus.
Decreased Proline Endopeptidase Activity in the Basal Ganglia in Huntington's Disease
Authors: Pittaway K. M. , Reynolds G. P. , Emson P. C.
Published: December 2006

Abstract: Soluble proline endopeptidase (EC 3.4.21.26) activity was measured by a fluorometric assay in eight human brain areas (caudate nucleus, lateral globus pallidus, medial globus pallidus, substantia nigrazona compacta, substantia nigra‐zona reticulata, frontal cortex‐Brodmann area 10, temporal cortex‐Brodmann area 38, and hippocampus), in 10 control and 10 Huntington's disease brains. An abnormally low activity (22% of control activity) was found in the caudate nucleus of Huntington's disease brains; significantly decreased activity was also detected in the lateral globus pallidus and medial globus pallidus (37% and 40% of control, respectively).
Developmental Differences Between Soluble and Membrane‐Bound Fractions of Choline‐O‐Acetyltransferase in Neonatal Mouse Brain
Authors: Benishin Christina G. , Carroll Paul T.
Published: December 2006

Abstract: Three fractions (one soluble and two membrane‐bound) of choline acetyltransferase (ChAT) isolated from a nerve ending fraction of mouse forebrain, which have previously been reported to differ in several biochemical and physical aspects, were also found to differ in their rates of postnatal development. At 2 days of age, the activity in all three fractions was very low. Sodium phosphate buffer‐soluble (cytoplasmic) ChAT activity increased significantly by 8 days of age, whereas the ChAT activity of the two membrane‐bound fractions (NaCl‐ and Triton‐soluble) did not increase until 13 days of age. These results suggested that the differences observed between the three fractions of ChAT prepared from mouse brain are not solely artifacts of the isolation procedure.
CL 218,872 Binding to Benzodiazepine Receptors in Rat Spinal Cord: Modulation by γ‐Aminobutyric Acid and Evidence for Receptor Heterogeneity
Authors: Villiger John W.
Published: December 2006

Abstract: The binding of the triazolopyridazine CL 218,872 to central benzodiazepine receptors identified with [3H]Ro 15–1788 was studied in extensively washed homogenates of rat spinal cord and cerebral cortex. CL 218,872 displacement curves were shallow in both spinal cord (nH= 0.67) and cortex (nH= 0.54), suggesting the presence of type 1 and type 2 benzodiazepine receptors in both tissues. CL 218,872 had lower affinity in spinal cord (IC50= 825 nM) than cortex (IC50= 152 nM), possibly reflecting the presence of fewer type 1 sites in the cord. Activating γ‐aminobutyric acid (GABA) receptors with 10 μM muscimol resulted in a two‐ to threefold increase in CL 218,872 affinity in both tissues without changes in the displacement curve slope. This indicates that GABA enhances CL 218,872 affinity for both type 1 and type 2 sites in both spinal cord and cerebral cortex.
Topographical Atlas of the Gangliosides of the Adult Human Brain
Authors: Kraĉun Ivica , Rösner Harald , Ćosović Cedomir , Stavljenić Ana
Published: December 2006

Abstract: Forty different brain samples, consisting of neocortical, archicortical, and paleocortical areas; telencephalic, diencephalic, and mesencephalic subcortical nuclei; and the cerebellum as well as some of the corresponding white matter bundles were analyzed with respect to total content of ganglioside‐sialic acid and the ganglioside pattern. The total content of gangliosides seems to depend mainly on the proportions of gray and white matter. Thus, neocortical areas, which are rich in gray matter, have a four‐ to fivefold higher ganglioside content (per milligram of protein) than white matter‐rich samples such as optic chiasm, capsula interna, or corpus callosum. White matter‐rich regions, although very heterogeneous in ganglioside composition, are further characterized by appreciable amounts of the myelin‐enriched GM4. In the neocortex a remarkable degree of regional pattern differences was revealed. In the frontal and parietal areas there is a moderate, and in the temporal region a strong preponderance of sialic acid bound to gangliosides of the a‐pathway (GD1a, GM1). In contrast, the occipital cortex favors the b‐pathway of ganglioside synthesis (GQ1b, GT1b, GD1b). A predominance of “b‐gangliosides” was found in all structures that are related to the visual system (optic chiasm, pulvinar‐thalamus, superior colliculi, visual cortex) as well as in the cerebellum and the nucleus ruber. All diencephalic nuclei tend to favor slightly “b‐gangliosides,” while the mesencephalic nuclei are very heterogeneous in their ganglioside composition. A preponderance of “a‐gangliosides” was found in the periamygdalar cortex, putamen, inferior colliculi, substantia nigra, frontal white matter, internal capsule, globus pallidus, basal nucleus of Meynert, and corpus callosum as well as in the frontal, parietal, and temporal cortices. An exceptional predominance of GM1 and GD1a was revealed for the hippocampal archicortex and the amygdala, suggesting a possible functional correlation to glutaminergic synaptic transmission.
Characterization of Na+‐Independent GABA and Flunitrazepam Binding Sites in Preparations of Synaptic Membranes and Postsynaptic Densities Isolated from Canine Cerebral Cortex and Cerebellum
Authors: Carlin Richard K. , Siekevitz Philip
Published: December 2006

Abstract: The binding of [3H]GABA and [3H]flunitrazepam was performed with synaptic membranes and postsynaptic densities (PSDs) isolated from canine cerebral cortex and cerebellum. Two GABA binding sites were found with cerebral cortex membranes but only one with cerebellar membranes. PSDs isolated from these showed only single binding sites, with cerebellar PSDs exhibiting lower KD values and a larger concentration of sites than did cerebral cortex PSDs. In the case of flunitrazepam, only one binding site was found for all four preparations, with cerebellar PSDs having twice the concentration of sites of cerebral PSDs. Photoaffinity labeling of the flunitrazepam receptor in PSDs resulted in the binding to a 51,000 Mr protein in both cases, with cerebellar PSDs again showing an increased concentration over that found in cerebral cortex PSDs. Based on this work, and on earlier work of ourselves and of others, we conclude that both populations of isolated PSDs contain inhibitory sites, but that the intact PSDs in both preparations are derived from Gray type I, probably excitatory, synapses, and that the inhibitory sites are found in the broken‐up material in the PSD fractions which are derived from Gray type II, probably inhibitory, synapses.
Regional Calcium Accumulation and Cation Shifts in Rat Brain by Kainate
Authors: Korf Jakob , Postema Folkert
Published: December 2006

Abstract: Following local application of kainic acid, changes in the contents of Na+, K+, Ca2+, and Mg2+ of the striatum, cerebellum, and hippocampus of the rat were observed at various times after surgery. Within 1 h the levels of K+ decreased 20% whereas the levels of Na+ and Ca2+ increased at least 50% and 20%, respectively. These changes persisted for more than 8 weeks. Ca2+ levels rose further, to more than 10‐fold during 8 weeks. The Mg2+ levels were slightly and only transiently decreased. Unilateral injections of kainate into the striatum affected the contents of these cations not only in this area, but also in the overlying cerebral cortex, the olfactory tubercle, and the ipsilateral substantia nigra. The Ca2+ increases were less when rats were kept on a diet deficient in Ca2+ and vitamin D. 45Ca2+, intravenously administered, accumulated significantly more in the kainate‐lesioned striatum and substantia nigra than in the homotopic contralateral areas. Electron microscopic examination of the localization of Ca2+ with the oxalate‐pyroantimonate technique showed the appearance of extracellularly located deposits and the accumulation of Ca2+ in (possibly degenerating) myelinated axons in kainate‐lesioned striata. This study provides evidence that calcification of cerebral tissue is closely associated with neurodegenerative processes and shows that kainate may serve as a tool to elucidate the mechanism of brain calcification. The results are discussed in relation to idiopathic calcinosis (striopallidodentate calcinosis, Fahr's disease).
Identification of Bradykinin in Mammalian Brain
Authors: Perry David C. , Snyder Solomon H.
Published: December 2006

Abstract: Bradykinin‐like activity was purified from acetic acid extracts of saline‐perfused rat brains by gel filtration chromatography and two reverse‐phase HPLC systems capable of resolving bradykinin from lysyl‐bradykinin and other bradykinin analogs and fragments. Addition of [3H]bradykinin to extracts permitted calculation of recoveries and monitoring of chromatographic fractions. Fractions were examined by radioimmunoassay using a potent and highly specific antiserum raised against bradykinin‐human albumin conjugates in rabbits. Bradykinin receptor‐active material was also measured by radioreceptor assay using guinea pig ileum, as well as by a bioassay with the estrous rat uterus. Active material chromatographed as authentic bradykinin in all systems. Levels of 0.6 pmol/g whole rat brain were detected, with eight times higher levels in the hypothalamus. Activity increased up to 10‐fold following treatment with trypsin; treatment with α‐chymotrypsin or angiotensin‐converting enzyme substantially reduced activity. Similar levels and distribution of bradykinin‐like activity were also detected in guinea pig brain extracts. These data substantiate the existence of authentic bradykinin in mammalian brain.
On the Status of Lysolecithin in Rat Cerebral Cortex During Ischemia
Authors: Sun Grace Y. , Foudin Laurie L.
Published: December 2006

Abstract: Lysolecithin (lysoglycerophosphocholine, LPC) was isolated from rat cerebral cortex and quantitatively analyzed at various times after postdecapitative ischemic treatment. In addition, different procedures for extraction and analysis of the LPC in brain were evaluated. Results indicated that LPC can be quantitatively extracted into the organic phase using the conventional extraction procedure with chloroform‐methanol (2:1, vol/ vol). However, care should be taken to avoid using strong acids, which can hydrolyze the alkenylether side chain of the plasmalogens, resulting in the release of 2‐acyl‐phospholipids. Quantitative GLC analysis using myris‐toyl‐LPC as internal standard revealed a level of 1.8 nmol LPC/mg protein in brain with acyl groups comprised mainly of 16:0, 18:0, and 18:1. The acyl group profile reflects that the LPC are derived mainly from phospho‐lipase A2 action. An increase of 46% in the LPC level was observed at 1 min after ischemic treatment, but this was followed by a steady decline. Ischemia induced an increase in the LPC species that are enriched in 18:0 and 18:1 fatty acids. The transient appearance of LPC during ischemia further suggests that this phospholipid is undergoing active turnover, possibly hydrolysis by the lysophospholipase. This mechanism of action may account, at least in part, for the increase in both saturated and unsaturated fatty acids during the early phase of the ischemic treatment.
Opiates Stimulate Low Km GTPase in Brain
Authors: Franklin Paul H. , Hoss Wayne
Published: December 2006

Abstract: Low Km GTP hydrolysis in rat brain is stimulated in a concentration‐dependent manner by the opiate alkaloid etorphine, and by the opioid peptide d‐Ala2‐leucine‐enkephalinamide. The opiate antagonist naloxone inhibits the maximal d‐Ala2‐leucine‐enkephalinamide stimulation of the GTPase, also with concentration dependency. The magnitude of maximally stimulated, opioid‐sensitive, GTP hydrolysis is differentially distributed across brain regions. Opioid‐stimulated GTPase may represent one means of identifying a specific type of opioid receptor.
Uptake, Metabolism, and Releasability of Ethyl Analogues of Homocholine by Rat Brain
Authors: Welner S. A. , Collier B.
Published: December 2006

Abstract: Ethyl analogues of homocholine were synthesized and used to describe further the specificities of the processes involved in choline uptake and acetylation and acetylcholine storage and release. Monoethylhomocholine, diethylhomocholine, and triethylhomocholine decreased the transport of choline into rat brain synaptosomes. The mono‐ and diethyl compounds were taken up into synaptosomes with similar affinity for the transport system as choline (5.8, 8.5, and 5.5 μM, respectively) but at a somewhat slower rate (11.3, 8.5, and 37.3 nmol/g original tissue/h, respectively); the triethyl analogue was not transported at the concentrations tested, which further defines the structural specificity of the transport system. l‐Carnitine did not affect the transport of the analogues. The in situ acetylation of mono‐ and diethyl‐homocholine by slices of rat cerebral cortex was measurable, but the in vitro acetylation by choline acetyl‐transferase solubilized from rat forebrain was not. Acetylation of the diethyl analogue by slices of cerebellar cortex was <20% of that by slices of cerebral cortex. Subcellular fractionation of cerebral slices showed that acetyldiethylhomocholine localized preferentially to the cytosolic rather than vesicular stores, indicating specificity of the mechanism responsible for the incorporation of acetylated product into the vesicles. The release of acetyldiethylhomocholine and of acetylcholine was tested from sliced brain that had been incubated with the precursors. Both esters were released spontaneously but stimulation with increased K+ concentration enhanced the release of acetylcholine without changing the release of acetyldiethylhomocholine, suggesting that evoked transmitter release occurred from a vesicular store.
Posttranslational Incorporation of [14C]Arginine into Rat Brain Proteins. Acceptor Changes During Development
Authors: Hallak Marta E. , Barra H. S. , Caputto R.
Published: December 2006

Abstract: Many of the cytosolic proteins of the rat brain appear to have the capacity to incorporate l‐[14C]arginine posttranslationally. Scanning of the electrophoretic pattern of the labeled proteins showed two main radioactive peaks: peak A, found in the region of proteins of MW above 200 kD, and peak B, found in the region of 33 kD. The ratio of peaks A/B tends to decrease with the age of the rats. Another zone of radioactivity has an apparent MW similar to that of albumin (approximately 66 kD). No differences were found between the effects of ionic strength and of inhibitors on the arginyl transferase of brain and those described for the transferases of other organs.
Low‐Na+ Medium Increases the Activity and the Phosphorylation of Tyrosine Hydroxylase in the Superior Cervical Ganglion of the Rat
Authors: Cahill Anne L. , Horwitz Joel , Perlman Robert L.
Published: December 2006

Abstract: Incubation of the rat superior cervical ganglion in Na+‐free or low‐Na+ medium increased the rate of synthesis of 3,4‐dihydroxyphenylalanine (DOPA) in the ganglion fourfold and caused a concomitant stable activation of tyrosine hydroxylase. DOPA synthesis was half‐maximal in medium containing about 20 mM Na+. Low‐Na+ medium also increased the incorporation of 32Pi into tyrosine hydroxylase; the dependence of tyrosine hydroxylase phosphorylation on the Na+ concentration resembled that of DOPA synthesis. The stimulatory effects of low‐Na+ medium on DOPA production and on tyrosine hydroxylase activity in vitro were dependent on extracellular Ca2+. The stimulation of DOPA synthesis in low‐Na+ medium was inhibited by methoxyverapamil, an inhibitor of Ca2+ uptake, and was partially blocked by tetrodotoxin, but it was not affected by the cholinergic antagonists hexamethonium and atropine. Ionomycin, a calcium ionophore, stimulated DOPA synthesis to about the same extent as low‐Na+ medium and also increased the incorporation of 32Pi into tyrosine hydroxylase. 8‐Bromo cyclic AMP (1 mM) also stimulated DOPA production in the ganglion, and this stimulation was more than additive with that produced by low‐Na+ medium. These data support the hypothesis that low‐Na+ medium stimulates DOPA synthesis by raising intracellular Ca2+, which then promotes the phosphorylation of tyrosine hydroxylase.
Immunohistochemical Localization of 2′,3′‐Cyclic Nucleotide 3′‐Phosphodiesterase and Myelin Basic Protein in the Central Nervous System of the Jimpy and the Normal Mouse
Authors: Mikoshiba Katsuhiko , Kohsaka Shinichi , Hayakawa Taketoshi , Takamatsu Ken , Tsukada Yasuzo
Published: December 2006

Abstract: We describe the immunohistochemical localization of 2′,3′‐cyclic nucleotide 3′‐phosphodiesterase (CNPase) and myelin basic protein (MBP) in CNS of the jimpy mutant mouse which is characterized by dys‐ and demyelination. In controls, the CNPase and MBP were localized exclusively in white matter in the CNS. The jimpy mutant mice were severely affected: A very weak reaction was observed in the white matter. Very few CNPase‐ and MBP‐positive myelin sheaths were observed, and some degradation products were also observed after reaction with antisera against both CNPase and MBP. The immunohistochemical reaction in the jimpy mice showed a similar localization in both CNPase and MBP.
Receptor‐Mediated Phosphorylation of Astroglial Intermediate Filament Proteins in Cultured Astroglia
Authors: McCarthy Ken D. , Prime Judith , Harmon Teven , Pollenz Richard
Published: December 2006

Abstract: Primary cultures of purified astroglia have been shown to exhibit a variety of membrane receptors that regulate intracellular cyclic AMP levels. The experiments described in this paper were completed to examine the effect of such receptor agonists on protein phosphorylation in intact astroglia. An analysis of 32P‐labelled proteins derived from whole cell extracts and separated via two‐dimensional gel electrophoresis indicated that increasing cyclic AMP levels in astroglia stimulated the phosphorylation of two distinct proteins that had apparent molecular weights/isoelectric points (pI) of 51K/ 6.0 and 57K/5.7. Similar experiments with cultured meningeal cells indicated that only the 57K/5.7 protein was phosphorylated in response to elevated levels of cyclic AMP. The 51K/6.0 protein was never observed in gels derived from meningeal cells. Immunoblot experiments indicated that the 51K/6.0 protein stained with antiserum to glial fibrillary acidic protein (GFAP) and the 57K/5.7 protein stained with antibodies to vimentin. Concentration‐effect studies indicate that these proteins are maximally phosphorylated at concentrations of receptor agonists that only slightly elevate cyclic AMP levels. All receptor agonists that have been shown to increase cyclic AMP levels appear similarly efficacious with respect to increasing the phosphorylation of the two proteins. These experiments suggest that the membrane receptors present on astroglia function, in part, to regulate phosphorylation of the intermediate filament proteins GFAP and vimentin.
Expression of A and B Types of Monoamine Oxidase in Differentiated Neuroblastoma Hybrid Cells
Authors: Nakano T. , Nagatsu T. , Higashida H.
Published: December 2006

Abstract: The total activities of monoamine oxidase (MAO) and the ratio of type B/type A activities were determined in mouse neuroblastoma N1E‐115 cells, and in NX31T and NG108‐15 hybrid cells derived from mouse neuroblastoma × rat sympathetic ganglion hybrid or mouse neuroblastoma × rat glioma hybrid cells. N1E‐115 and NX31T cells possessed type A activities exclusively, although NG108‐15 cells showed both type A (65–90%) and type B (10–35%) MAO activities. The activity of type A MAO in NX31T and N1E‐115 cells was relatively constant during culturing periods in the presence or absence of dibutyryl cyclic AMP (Bt2cAMP), whereas total MAO activity and the ratio of type B MAO/type A MAO in NG108‐15 cells increased as a function of culture periods. Prostaglandin E1 (PGE1) and theophylline, the best known combination to increase intracellular cyclic AMP content of NG108‐15 cells, caused similar increases of MAO and of the type B/type A ratio in NG108‐15 cells. The results suggest that MAO activity and expression of MAO B activity are regulated in NG108‐15 cells in a cyclic AMP‐dependent manner.
Vasoactive Intestinal Polypeptide Receptors Linked to an Adenylate Cyclase, and Their Relationship with Biogenic Amine‐ and Somatostatin‐Sensitive Adenylate Cyclases on Central Neuronal and Glial Cells in Primary Cultures
Authors: Chneiweiss H. , Glowinski J. , Prémont J.
Published: December 2006

Abstract: The presence of vasoactive intestinal polypeptide (VIP) receptors coupled to an adenylate cyclase was demonstrated on membranes of neurons or glial cells grown in primary cultures originating from the cerebral cortex, striatum, and mesencephalon of mouse embryos. A biphasic pattern of activation was observed in all these cell types, involving distinct high‐ and low‐apparent‐affinity mechanisms. The absence of additive effects of VIP and 3,4‐dihydroxyphenylethylamine (DA, dopamine), isoproterenol (ISO), and 5‐hydroxytryptamine (5‐HT, serotonin) suggests that the peptide receptors are colocated with each of the corresponding amine receptors on neuronal membranes of the three structures studied. The nonadditivity between the VIP‐ and ISO‐induced responses on cortical and striatal glial membranes reveals as well a colocation of VIP and β‐adrenergic‐sensitive adenylate cyclases on the same cells. A subpopulation of mesencephalic glia could possess only one of the two types of receptors, as a partial additivity of the VIP and ISO responses was seen. In addition, VIP modified the characteristics of the somatostatin inhibitory effect on adenylate cyclase activity of neuronal membranes from the cerebral cortex and striatum but not from those of the mesencephalon. On striatal and mesencephalic glial membranes the somatostatin inhibitory effect was observed only in the presence of VIP. However, as previously seen with ISO, the presence of VIP did not allow the appearance of a somatostatin inhibitory response on cortical glial membranes. This suggests that cortical glia are devoid of somatostatin receptors.
Effects of l‐DOPA on the Concentrations of Free and Sulfoconjugated Cathecholamines in Plasma, Cerebrospinal Fluid, Urine, and Central and Peripheral Nervous System Tissues of the Rat
Authors: Buu Nguyen T. , Duhaime Johanne , Kuchel Otto
Published: December 2006

Abstract: The effects of subeutaneous injection of l‐β‐3,4‐dihydroxyphenylalanine (l‐DOPA) on the concentrations of the catecholamines and catecholamine sulfates in the central and peripheral nervous Systems of the rat were studied. The results showed that free 3,4‐dihydroxyphenylethylamine (DA, dopamine) increased rapidly and markedly in the hypothalamus and striatum after l‐DOPA but DA sulfate did not change. Increased concentrations of DA sulfate were detected in the CSF and in the plasma, where it reached a concentration of 130.8 ± 12.8 ng/ml at 2 h, seven times the level of free DA (19.1 ± 2.9 ng/ml). In the kidney the ratio of DA sulfate to free DA was reversed in favor of free DA. Urine samples of l‐DOPA‐treated rats showed a higher increase of free DA than DA sulfate, but free norepinephrine (NE) and NE sulfate remained unchanged. Concentrations of free DA and free NE in the adrenal glands of l‐DOPA‐treated rats showed no change. Adrenal DA sulfate and NE sulfate were not detectable in the control and l‐DOPA‐treated rats, suggesting that the adrenal glands lack the capacity to take up or store catecholamines and their sulfate counterparts from the plasma.
Effects of Systemically Administered Lithium on Phosphoinositide Metabolism in Rat Brain, Kidney, and Testis
Authors: Sherman William R. , Munsell Ling Y. , Gish Beverly G. , Honchar Michael P.
Published: December 2006

Abstract: A single subcutaneous dose of 10 mEq/kg LiCl gives rise to an increase in the cerebral cortex level of myo‐inositol‐1‐P (I1P) that closely follows cortical lithium levels and, at maximum, is 40‐fold above the control value. Kidney and testis show smaller increases in I1P level following LiCl administration. The I1P level is still sixfold greater than that of untreated rat cortex 72 h later. In cortex, parallel increases also occur in myo‐inositol‐4‐P (I4P) and myo‐inositol 1,2‐cyclic‐P (cI1, 2P), whereas myo‐inositol‐5‐P (I5P) remains unchanged. The cortical increases in I1P and I4P levels are partially reversed by administering 150 mg/kg of atropine 22 h after the LiCl, treatment that does not affect cI1, 2P. When doses of LiCl from 2 to 17 mEq/kg are given, the cerebral cortex levels of I1P and myo‐inositol, measured 24 h later, are found to reach a plateau at about 9 mEq/kg of LiCl, whereas cortical lithium levels continued to increase with greater LiCl doses. Levels of ail three of the brain phosphoinositides are unchanged by the 10 mEq/kg LiCl dose, as is the uptake of 32Pi into these lipids. Chronic dietary administration of LiCl for 22 days showed that the effects of lithium on I1P and myo‐inositol levels persist for that period. Over the course of the chronic administration of the lithium, levels of I1P, myo‐inositol, and of lithium in cortex remained significantly correlated. We believe that these increases in inositol phosphates result from endogenous phosphoinositide metabolism in cerebral cortex and that lithium is capable of modulating that metabolism by reducing cellular myo‐inositol levels. The size of the effect is a function of both lithium dose and the degree of stimulation of receptor‐linked phosphoinositide metabolism. This property of lithium may explain part of its ability to moderate the symptoms of mania. Our chronic study suggests that prolonged administration of LiCl does not resuit in compensatory changes in myo‐inositol‐1‐P synthase or myo‐inositol‐1‐phosphatase.
Expression of A1 Adenosine Receptors Modulating Dopamine‐Dependent Cyclic AMP Accumulation in the Chick Embryo Retina
Authors: de Carvalho R. Paes , de Mello F. G.
Published: December 2006

Abstract: Dopamine and 2‐chloroadenosine independently promoted the accumulation of cyclic AMP in retinas from 16‐day‐old chick embryos. The two compounds added together either in saturating or subsaturating concentrations were not additive for the accumulation of the cyclic nucleotide in the tissue. This fact was shown to be due to the existence of an adenosine receptor that mediates the inhibition of the dopamine‐dependent cyclic AMP accumulation in the retina. Adenosine inhibited, in a dose‐dependent fashion, the accumulation of cyclic AMP induced by dopamine in 12‐day‐old chick embryo retinas, with an IC50 of approximately 1 μM. This effect was not blocked by dipyridamole. N6‐ (l‐Phenylisopropyl)adenosine, (l‐PIA) was the most potent adenosine analog tested, showing an IC50 of 0.1 μM which was two orders of magnitude lower than its stereoisomer cf‐PIA (10 μM). The maximal inhibition of the dopamine‐elicited cyclic AMP accumulation by adenosine and related analogs was 70%. The inhibitory effect promoted by adenosine was blocked by 3‐isobutyl‐l‐methylxanthine (IBMX) or by adenosine deaminase. Adenine was not effective, whereas ATP and AMP promoted the inhibition of the dopamine effect only at very high concentrations. Apomorphine was only 30% as effective as dopamine in promoting the cyclic AMP accumulation in retinas from 11‐ to 12‐day‐old embryos and 2‐chloroadenosine did not interfere with the apomorphine‐mediated shift in cyclic AMP levels. In the retinas from 5‐day‐old posthatched chickens dopamine and apomorphine were equally effective in eliciting the accumulation of cyclic AMP. In this case, 2‐chloroadenosine did not interfere with the response elicited by either one of the compounds. The data suggest the existence of two populations of D1 dopamine receptors in the chick embryo retina—one that is negatively modulated by adenosine and another that is not under purinergic control.
Drug Potencies on Partially Purified Brain D2 Dopamine Receptors
Authors: Madras Bertha K. , Seeman Philip
Published: December 2006

Abstract: To examine the sensitivities of partially purified dopamine receptors to various dopaminergic agonists and antagonists, canine brain striatum dopamine receptors were enriched by isoelectric focusing. The digitonin‐solubilized receptors were prelabelled with [3H]spiperone and focused for two time periods. After 5 h (incomplete focusing), radioactive peaks were detected at pH 6 and 9–11. Only the pH 6 peak revealed drug sensitivities expected of D2 receptors. Receptor recovery of the pH 6 peak was 79% with purification being sevenfold. After focusing overnight to equilibrium, the pH 6 peak further separated into peaks at pH 4.6 and 6.8. The receptor was identified only in the pH 4.6 fraction. The recovery of receptors in the pH 4.6 peak was low (10%), indicating little enrichment of the receptor. The rank order of binding of neuroleptics and dopamine agonists to the purified material was similar to that of the original preparation of soluble receptors. Dopamine did not bind to the purified pH 4.6 fraction unless the phosphate buffer (used during focusing) was replaced with Tris buffer. The absence of receptors in the pH 6.8 and pH 10 fractions, although both contained prelabeled [3H]spiperone, indicates the importance of testing agonists and antagonists on each fraction at each step in purification.
Glycosaminoglycans of Rat Cerebellum: I. Quantitative Analysis of the Main Constituents at Postnatal Day 6
Authors: Werz W. , Fischer G. , Schachner M.
Published: December 2006

Abstract: Isolated glycosaminoglycans (GAGs) were quantified biochemically in the cerebella of 6‐day‐old rats. 14C‐Labeled hyaluronic acid (HA) and chondroitin‐4‐sulfate (C‐4‐S), added prior to isolation of GAGs from tissue, served as internal standards to allow correction for unknown losses during the purification procedure and exact quantification of GAGs in the intact tissue. Three main constituents—HA, chondroitin sulfate (CS), and heparan sulfate (HS)—were found at concentrations of 1.82, 1.52, and 0.76 μg/mg protein amounting to 44%, 37%, and 19% of the total GAG fraction, respectively. Incorporation of [3H]glucosamine precursor into GAGs was higher for HS (56% of incorporated precursor) and lower for HA (29%) and CS (15%). The specific activities of individual GAGs were 64.7 nCi/μg for HS, 14.2 for HA, and 8.3 for CS.
Kinetics of Regional Blood‐Brain Barrier Glucose Transport and Cerebral Blood Flow Determined with the Carotid Injection Technique in Conscious Rats
Authors: Braun Leon D. , Miller Leonard P. , Pardridge William M. , Oldendorf William H.
Published: December 2006

Abstract: Anesthetics, particularly barbiturates, have depressive effects on cerebral blood flow and metabolism and likely have similar effects on blood‐brain barrier (BBB) transport. In previous studies utilizing the carotid injection technique, it was necessary to anesthetize the animals prior to performing the experiment. The carotid injection technique was modified by catheter implantation in the external carotid artery at the bifurcation of the common carotid artery. The technique was used to determine cerebral blood flow, the Km, Vmax, and KD of glucose transport in hippocampus, caudate, cortex, and thalamus‐hypothalamus in conscious rats. Blood flow increased two to three times from that seen in the anesthetized rat. The Km in the four regions ranged between 6.5 and 9.2 mM, the Vmax ranged between 1.15 and 2.07 μmol/min/g, and the KD ranged between 0.015 and 0.035 ml/min/g. The Km and KD in the conscious rat did not differ from the values seen in the barbiturate anesthetized rat. The Vmax, on the other hand, increased two‐ to three‐fold from that seen in the anesthetized rat and was nearly proportional to the increase in blood flow seen in the conscious rat. The development of the external carotid catheter technique now allows for determination of BBB substrate transport in conscious animals.
Influence of δ‐Opioid Receptors on Production of Labeled Methionine5‐Enkephalin in Murine Neuroblastoma Cells
Authors: Gilbert Judith A. , Richelson Elliott
Published: December 2006

Abstract: Synthesis of methionine5‐enkephalin by intact cells of murine neuroblastoma clone N1E‐115 has been demonstrated both immunocytochemically and biochemically. In addition, N1E‐115 cells possess homogeneous enkephalin (δ) receptors which inhibit prostaglandin E1‐ induced intracellular cyclic AMP formation. An assay was developed for measuring de novo synthesis of methionine5‐enkephalin by pulsing cells in culture with radioactive methionine and isolating this pentapeptide to radiochemical purity by a procedure that included immunoaffinity chromatography specific for oxidized methionine5‐enkephalin. This assay indicated that production of radiolabeled‐methionine5‐enkephalin was increased upon lengthy exposure of intact N1E‐115 cells in the late logarithmic phase of growth to a nonproteolyz‐able analog of methionine5‐enkephalin. This increase in synthesis of intracellular methionine5‐enkephalin relative to control cells was prevented by prior incubation of the clone with naloxone, indicating that the response was mediated by the δ receptor.
Effect of Electrical Stimulation on Phosphoinositide Metabolism in Rat Sciatic Nerve In Vivo
Authors: Goswami S. K. , Gould R. M.
Published: December 2006

Abstract: The metabolism of phosphoinositides in rat sciatic nerves in vivo during electrical stimulation was studied. Nerves were prelabeled by injection of [2‐3H]‐myo‐inositol alone for periods of 2 and 20 h or together with [32P]orthophosphate for 2 h and then electrically stimulated (100 Hz) for 5 or 20 min. Contralateral unstimulated nerve served as the control. When tritiated myo‐inositol was used alone for prelabeling the nerves, approximately 6% and 14% of the label was incorporated into lipids after 2 h and 20 h, respectively. Both 5 and 20 min of electrical stimulation caused an insignificant change in the percentage of radioactivity recovered in lipids from the nerves prelabeled with either myo‐inositol or with a mixture of myo‐inositol and phosphate. The proportion of label associated with phosphoinositides of nerves prelabeled with myo‐inositol for both 2 h and 20 h showed an increase in phosphatidyl‐inositol‐4‐phosphate at the expense of phosphatidylinositol in stimulated nerves. Similar results were obtained with nerves prelabeled for 2 h with a mixture of [32P]orthophosphate and [2‐3H]myo‐inositol. No significant changes in the radioactivity associated with water‐soluble inositol phosphates were found in stimulated versus control nerves.