bluuebunny/crossref_metadata_2025 huggingface dataset:
Cholinergic Stimulation of Inositol Phosphate Formation in Bovine Adrenal Chromaffin Cells: Distinct Nicotinic and Muscarinic Mechanisms
Authors: Eberhard D. A. , Holz R. W.
Published: October 2006

Abstract: The ability of cholinergic agonists to activate phospholipase C in bovine adrenal chromaffin cells was examined by assaying the production of inositol phosphates in cells prelabeled with [3H]inositol. We found that both nicotinic and muscarinic agonists increased the accumulation of [3H]inositol phosphates (mainly inositol monophos‐phate) and that the effects mediated by the two types of receptors were independent of each other. The production of inositol phosphates by nicotinic stimulation required extracellular Ca2+ and was maximal at 0.2 mMCa2+. Increasing extracellular Ca2+ from 0.22 to 2.2 mM increased the sensitivity of inositol phosphates formation to stimulation by submaximal concentrations of 1,1‐dimethyl‐4‐phenyl‐piperazinium iodide (DMPP) but did not enhance the response to muscarine. Elevated K+ also stimulated Ca2+‐dependent [3H]inositol phosphate production, presumably by a non‐receptor‐mediated mechanism. The Ca2+ channel antagonists D600 and nifedipine inhibited the effects of DMPP and elevated K+ to a greater extent than that of muscarine. Ca2+ (0.3–10 μM) directly stimulated the release of inositol phosphates from digitonin‐permeabilized cells that had been prelabeled with [3H]inositol. Thus, cholinergic stimulation of bovine adrenal chromaffin cells results in the activation of phospholipase C by distinct muscarinic and nicotinic mechanisms. Nicotinic receptor stimulation and elevated K+ probably increased the accumulation of inositol phosphates through Ca2+ influx and a rise in cytosolic Ca2+. Because Ba2+ caused catechol‐amine secretion but did not enhance the formation of inositol phosphates, phospholipase C activation is not required for exocytosis. However, diglyceride and wyo‐inositol 1,4,5‐trisphosphate produced during cholinergic stimulation of chromaffin cells may modulate secretion and other cellular processes by activating protein kinase C and/or releasing Ca2+ from intracellular stores.
Monoclonal Antibodies to Substance P: Production, Characterization of Their Fine Specificities, and Use in Immunocytochemistry
Authors: Couraud J. Y. , Frobert Y. , Conrath M. , Renzi D. , Grassi J. , Drapeau G. , Regoli D. , Pradelies P.
Published: October 2006

Five hybrid clones secreting antibodies to the neuropeptide substance P have been obtained by somatic cell fusion of mouse myeloma cells with splenocytes from immunized mice of the Biozzi strain. To perform rapid and sensitive screening tests as well as to study the fine specificities of each monoclonal antibody, we developed a new enzyme immunoassay of substance P using acetylcholines‐ terase as label. All five monoclonal antibodies were directed to the C‐terminal pentapeptide of substance P, especially to the Phe7 residue. They cross‐reacted with neurokinin A and to some extent with neurokinin B but not with other non‐tachykinin mammalian peptides. One monoclonal antibody (SP 14) was used for immunocytochemical experiments in the rat spinal cord and spinal ganglion, both at the light and electron microscopic levels. A strong specific neurokinin‐like immuno eactivity was observed in cell bodies, nerve fibers, and terminals, with a very low background staining. Finally, the affinities of several analogues of substance P for SP14 manoclonal antibody were shown to be correlated with their biological activities, as measured by their hypotensive effects in vivo. These findings suggested a strong structural resemblance between the combining site of the antibody and that of the physiological substance P receptor.
Mediation of Norepinephrine‐Stimulated Cyclic AMP Accumulation by Adrenergic Receptors in Hypothalamic and Preoptic Area Slices: Effects of Estradiol
Authors: Etgen Anne M. , Petitti Nicolas
Published: October 2006

Adrenergic receptor agonists and antagonists were employed to establish (a) which receptor subtypes mediate the cyclic AMP response to norepinephrinc in hypo‐ thalamic and preoptic area slices from gonadectomized female rats and (b) which receptor subtypes might be modulated by the steroid hormone estradiol. Slice cyclic AMP levels were elevated by the β receptor agonist isoproterenol, but not by α1 (phenylephrine, methoxamine) or α2 (clonidine) agonists. However, the a agonist phenylephrine potentiated the effect of the β agonist isoproterenol on slice cyclic AMP accumulation. In slices from rats given no hormone treatment, the β antagonist propranolol inhibited norepinephrine‐stimulated cyclic AMP production, while the α1 antagonist prazosin was without effect. In contrast, the cyclic AMP response to norepinephrine in slices from estradiol‐treated rats was blocked more effectively by prazosin than by propranolol Estradiol treatment also attenuated the production of cyclic AMP by the β agonist isoproterenol. The data suggest (a) that norepinephrine induction of cyclic AMP accumulation in hypothalamic and preoptic area slices is med ated by β receptors and potentiated by a receptor activation and (b) that estradiol depresses β and increases α1 eceptor function in slices from brain regions associated with reproductive physiology.
Mutations Affecting Acetylcholine Levels in the Nematode Caenorhabditis elegans
Authors: Hosono Ryuji , Sassa Toshihiro , Kuno Sigeru
Published: October 2006

Gene cha‐1 · unc‐17 of the nematode Caenorhabditis elegans is a complex gene, consisting of at least two complementation groups. One part (cha‐1 region) of the gene encodes the enzyme choline acetyltransferase (ChAT), but the function of the other part (unc‐17 region) is still unclear. We measured the ChAT activity and ACh levels of the cha‐1 and unc‐17 complex gene mutants. We show here that alterations in ACh levels, rather than the ChAT activity, reflect abnormal phenotypes accompanying cha‐1 · unc‐17 mutations, that is, the decreased ACh levels in cha‐1 mutations and abnormal accumulation in unc‐17 mutations. Our results suggest that the unc‐17 region may encode functions necessary for storage and/or release of ACh at the presynaptic level.
Inositol Phospholipid Hydrolysis by Rat Sciatic Nerve Phospholipase C
Authors: Natarajan V. , Schmid Harald H. O.
Published: October 2006

Rat sciatic nerve cytosol contains a phosphodiesterase of the phospholipase C type that catalyzes the hydrolysis of inositol phospholipids, with preferences of phospha‐tidylinositol 4′‐phosphate (PIP) > phosphatidylinositol (PI) « phosphatidylinositol 4′, 5′‐bisphosphate (PIP2), at a pH optimum of 5.5–6.0 and at maximum rates of 55, 13, and 0.7 nmol/min/mg protein, respectively. Analysis of reaction products by TLC and formate exchange chromatography shows that inositol 1, 2‐cyclic phosphate (83%) and diacylglycerol are the major products of PI hydrolysis. [32P]PIP hydrolysis yields inositol bisphosphate, inositol phosphate, and inorganic phosphate, indicating the presence of phosphodiesterase, phosphomonoesterase, and/or inositol phosphate phosphatase activities in nerve cytosol. Phosphodiesterase activity is Ca2+‐dependent and completely inhibited by EGTA, but phosphomonoesterase activity is independent of divalent cations or chelating agents. Phosphatidylcholine (PC) and lysophosphatidylcholine (ly‐soPC) inhibit PI hydrolysis. They stimulate PIP and PIP2 hydrolysis up to equimolar concentrations, but are inhibitory at higher concentrations. Both diacylglycerols and free fatty acids stimulate PI hydrolysis and counteract its inhibition by PC and lysoPC. PIP2 is a poor substrate for the cytosolic phospholipase C and strongly inhibits hydrolysis of PI. However, it enhances PIP hydrolysis up to an equimolar concentration.
Triethyllead Inhibits γ‐Aminobutyric Acid Binding to Uptake Sites in Synaptosomal Membranes
Authors: Seidman Brenda C. , Olsen R. W. , Verity M. Anthony
Published: October 2006

Triethyllead (TEL), the active metabolite of tetraethyllead, was shown previously to inhibit selectively high‐affinity Na+‐dependent uptake of γ‐aminobutyric acid (GABA) into cerebrocortical synaptosomes. Such inhibition was not related to the Na+ gradient, Na+,K+‐ATPase activity, [Cl‐], or energy charge. We report here that TEL inhibits GABA binding to the presynaptic transporter involved in Na+‐dependent uptake. Scatchard plot analysis of Na+‐dependent [3H]GABA binding to a highly purified synaptic plasma membrane preparation revealed that 25 μM TEL reduced the Bmax by 44%, leaving the KD unchanged. This binding was reversible and predominantly involved membrane uptake sites, as characterized by pharmacological specificity to GABA ligands. Approximately 85% of specific GABA binding was considered membrane uptake site binding, as indicated by sensitivity to nipecotic acid and diaminobutyric acid, with relative insensitivity to muscimol, bicuculline methiodide, baclofen, and β‐alanine. With respect to previous data, these findings suggest that TEL inhibits Na+‐sensitive high‐affinity GABA uptake by interfering with GABA binding to its presynaptic transporter.
Monoclonal Antibodies Against Glutaraldehyde‐Conjugated Dopamine
Authors: Chagnaud Jean‐Luc , Mons Nicole , Tuffet Sophie , Grandier‐Vazeilles Xavier , Geffard Michel
Published: October 2006

Four mice were immunized with dopamine (DA)—glutaraldehyde (G)—protein conjugates over a period of 8–10 weeks. Polyclonal antisera, obtained at various intervals, were tested using an enzyme‐linked immuno‐sorbent assay (ELISA). All had anti‐conjugated DA antibodies. As soon as good antibody affinity was detected between 10‐10 and 10‐6M, the mouse yielding the highest apparent affinity was killed, and the spleen was dissected out. Hybridomas were obtained from spleen cells fused with SP2/O/Ag myeloma cells. Supernatant culture media of hybridomas were tested for the presence of anti‐conjugated DA antibodies with the ELISA method. Selected hybridomas giving good antibody affinity and specificity were then cloned by the limiting dilution technique. The resulting supernatant culture media were again tested by ELISA. Clones that gave a high antibody affinity (10‐10‐10‐8M) for G‐conjugated DA were used for histochemical localization of DA in rat brain. G‐fixed rat brains were sectioned from the telencephalon to the mesencephalon, reduced with sodium borohydride, and prepared for peroxidase‐antiperoxidase immunocytochemistry using supernatant (diluted 1:100) or ascites fluid (diluted 1:50,000). Dense networks of very fine fibers were observed in the striatum, septum, and cortex. Numerous immunoreactive cell bodies were found in the ventral tegmental area, the substantia nigra, the hypothalamus, and the dorsal raphe. The ELISA tests and adsorption controls suggested that the monoclonal antibody allowed highly specific detection of DA in tissues.
A Dopaminergic Cell Line Variant Resistant to the Neurotoxin 1‐Methyl‐4‐Phenyl‐1,2,3,6‐Tetrahydropyridine
Authors: Denton Timothy , Howard Bruce D.
Published: October 2006

1‐Methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) is known to cause parkinsonism by killing dopaminergic neurons; the toxic substance is a metabolite, 1‐methyl‐4‐phenylpyridinium ion (MPP+). PC12 cells, which are dopaminergic, are killed in culture by MPTP and MPP+ but at concentrations much higher than that required to kill affected neurons in vivo. However, at low concentrations (10–100 μM), MPP+ caused an increased production of lactate by PC12 cells. MPP+‐treated PC12 cells exhibited decreased mitochondrial respiration. Mitochondria from the treated cells respired normally in the presence of added succinate but not β‐hydroxybutyrate, a finding indicating that MPP+ inhibits the oxidation of some substrates selectively. MPP+ was more effective in killing the cells when glycolysis was reduced with 2‐deoxyglucose or by lowering the glucose content of the culture medium. Under these conditions, MPP+ inhibited ATP synthesis and depleted cellular stores of ATP. A PC12 variant that is even more resistant to MPTP and MPP+ than are wild‐type cells has been isolated. The MPTP‐resistant variant is also more resistant to the lethal effects of oligomycin, antimycin A, and rotenone. This variant exhibited altered lactate production and mitochondrial respiration. It is suggested that some brain neurons that accumulate MPP+ without being killed by it may also have an energy metabolism somewhat different from that of more sensitive neurons.
Acute and Long‐Term Effects of Chlorpromazine on Glutamine Synthetase and Glutaminase in Rat Brain
Authors: Chandrakala M. V. , Marcus Sara Rani , Nadiger H. A. , Sadasivudu B.
Published: October 2006

Abstract: The effect of administration of chlorpromazine on the activity of glutamine synthetase and glutaminase and the content of glutamate and γ‐aminobutyric acid (GABA) in different regions of rat brain was studied in an investigation of the possible role of these amino acids in the lowering of the seizure threshold following prolonged administration of chlorpromazine. Chlorpromazine was administered at a dose of 20 mg/kg of body weight s.c. For the acute study, the animals were killed 20 min after a single injection. For the long‐term study, the animals were treated every day with the same dose for 21 days and were killed 20 min after the last injection. The results showed an increase in glutamate level in each brain region investigated following long‐term administration, but only in the cerebral cortex after a single dose. GABA levels showed an increase in the brainstem only in acute experiments. Glutamine synthetase activity was increased in all three regions after a single dose and only in cerebral cortex after long‐term administration. Glutaminase activity showed a decrease in cerebral cortex only after long‐term administration of the drug. These results suggest the possible occurrence of a state of increased excitability in the brain as a result of long‐term administration of chlorpromazine, thus contributing to the known complication of seizures.
Dihydropyridine Modulation of the Chromaffin Cell Secretory Response
Authors: Ladona M. G. , Aunis D. , Gandía L. , García A. G.
Published: October 2006

Abstract: Prolonged perfusion of cat adrenal glands with Krebs‐bicarbonate solutions containing nicotine, musca‐rine, or excess K rapidly increased the rate of catecholamine output proportional to the concentrations of secretagogue used. The secretory responses to nicotine or high K reached a peak and declined to almost basal rates of secretion after about 10 min of stimulation. The dihydropyridine Ca channel agonist Bay K 8644 potentiated markedly the secretory responses to 1 μM nicotine and to 17.7 mM K but not to higher concentrations of these secretagogues. The musca‐rinic response did not decrease with time and was modestly potentiated by Bay K 8644. Similar curves were obtained with 17.7 mM K plus Bay K 8644 and with 59 mMK alone. CGP28392, another agonist, was about 10 times less potent than Bay K 8644 in potentiating the secretory responses to 17.7 mM K. Bay K 8644 also potentiated the release of I3H]noradrenaline evoked by stimulation of cultured bovine adrenal chromaffin cells with 17.7 mMK or 2μM nicotine but not with higher concentrations of K or nicotine. Dihydropyridine Ca channel antagonists reversed the effects of Bay K 8644 with the following order of potency: niludi‐pine ≥ nifedipine = nimodipine ≥ nitrendipine. The secretory rates from intact chromaffin cells treated with the Ca ionophores X537A or A23187, or those evoked by Ca‐EGTA buffers from digitonin‐permeabilized cells, were not affected by Bay K 8644. These results are compatible with the following conclusions: (1) Bay K 8644 selectively potentiates catecholamine secretory responses mediated through the activation of voltage‐sensitive Ca channels; (2) during nicotine or high‐K stimulation, Ca gains access to the cell interior through a common permeability pathway, the Ca channel; and (3) dihydropyridine Ca agonists and antagonists act on a common site on the chromaffin cell plasma membrane, perhaps a dihydropyridine receptor near the voltage‐dependent Ca channel and regulating it.
In Vivo Assessment of Dopamine and Norepinephrine Release in Rat Neocortex: Gas Chromatography‐Mass Spectrometry Measurement of 3‐Methoxytyramine and Normetanephrine
Authors: Wood Paul L. , Kim Helen S. , Altar C. Anthony
Published: October 2006

Abstract: A new method with the sensitivity and specificity required to measure regional levels of 3‐methoxytyramine (3‐MT) and normetanephrine (NMN) in the rat cortex is described. The method utilizes a liquid ion exchanger to isolate the parent amines, dopamine (DA) and norepinephrine (NE), along with their methylated metabolites. These samples are derivatized and analyzed by negative ion gas chro‐matography‐mass spectrometry. Using this method, we examined a number of drug actions on steady‐state levels as well as pargyline‐induced increases in 3‐MT and NMN. In the prefrontal cortex, cingulate cortex, striatum, and olfactory tubercle, nomifensine was found to increase 3‐MT steady‐state levels and accumulation rates. Similar actions of this drug were observed in the cingulate and prefrontal cortices with NMN. In contrast, clonidine decreased cortical NMN levels and accumulation. A unique action was observed with haloperidol, in that both 3‐MT levels and accumulation after pargyline were increased in the nigrostriatal and mesolimbic dopaminergic projections, whereas only the accumulation rates were accelerated in the mesocortical projections. In summary, our data indicate that this new assay is a useful approach for the in vivo evaluation of DA and NE release in cortical regions of the rat. This approach is unique in that no surgery, restraint, or anesthetic is required, thereby permitting more complicated experimental paradigms to be utilized.
Purification and Properties of Prostaglandin H‐E Isomerase from the Cytosol of Human Brain: Identification as Anionic Forms of Glutathione S‐Transferase
Authors: Ogorochi Toshiya , Ujihara Mayumi , Narumiya Shuh
Published: October 2006

Abstract: Prostaglandin H‐E isomerase (EC 5.3.99.3) was purified from human brain cytosol. Purification was by ammonium sulfate fractionation, diethylaminoethyl‐Sephar‐ose chromatography, gel filtration on a BioGel P‐100 column, GSH‐agarose chromatography, and MonoQ chromatography. The activity was eluted in two peaks from the MonoQ column, which were designated peaks 1 and 2. The molecular weights of peaks 1 and 2, determined by gel filtration, were 42,000 and 44,000, respectively. On sodium dodecyl sulfate‐polyacrylamide gel electrophoresis, peak 1 showed two bands at the molecular weights of 24,500 and 25,000, and peak 2 showed a single band at the molecular weight of 25,000, results suggesting that both were dimeric proteins. The pI values of both enzymes were ∼5.4. The enzymes catalyzed selective conversion of prostaglandin H2 to prostaglandin E2. The Km values for prostaglandin H2 of peaks 1 and 2 were 147 and 308 μM, respectively, and the Vmax values were 380 and 720 nmol/min/mg of protein, respectively. GSH was required for the catalysis of both enzymes, and no other sulfhydryl compounds could support the reaction. A part of glutathione S‐transferase (EC 2.5.1.18) was copurified with peaks 1 and 2 of prostaglandin H‐E isomerase. Prostaglandin H‐E isomerase activity of peak 2 enzyme was competitively inhibited by 1‐chloro‐2,4‐dinitrobenzene, a substrate of glutathione S‐transferase. These results suggested that prostaglandin H‐E isomerases in human brain cytosol were identical with anionic forms of glutathione S‐transferase.
Effect of Anticonvulsant Drugs on 7‐Hydroxybutyrate Release from Hippocampal Slices: Inhibition by Valproate and Ethosuximide
Authors: Vayer Philippe , Charlier Brigitte , Mandel Paul , Maitre Michel
Published: October 2006

Abstract: The effects of some anticonvulsant drugs have been investigated on γ‐hydroxybutyrate release from rat hippocampal and striatal slices. Sodium valproate and ethosuximide inhibited the depolarization‐evoked release of γ‐hydroxybutyrate induced by 40 mM K+. The IC50 values for these two drugs are in the concentration range of valproate and ethosuximide that exists in rat brain after administration of anticonvulsant doses to the animals. Trimethadione and pentobarbital are without significant effects. It can be concluded that the inhibition of γ‐hydroxybutyrate release, particularly that observed for hippocampus, might explain the protective effect of valproate and ethosuximide on γ‐hydroxybutyrate‐induced seizures and perhaps on other kinds of epileptoid phenomenon.
Myelin Proteolipid Protein Gene Expression in Jimpy and Jimpymsd Mice
Authors: Minnetta  , Gardinier V. , Macklin Wendy B.
Published: October 2006

Abstract: Proteolipid protein (PLP) gene expression was studied in the dysmyelinating mouse mutant jimpymsd (jpmsd; myelin synthesis deficient) and compared with that in wild‐type mice and the allelic mutant, jimpy (jp). Southern analyses of genomic DNA from jpmsd mice revealed no major rearrangements of the PLP gene relative to the wild‐type mouse PLP gene. PLP‐specific mRNA levels were significantly reduced in these mutant mice, although both the 3.2‐ and 2.4‐kilobase PLP‐specific mRNAs were seen. Also, no size differences in either PLP or DM20 mRNAs were found by S1 nuclease assays of brain RNA from either jpmsd or wild‐type mice. Both PLP and DM20 protein were detectable at low levels in jpmsd brain homoge‐nates, and these proteins comigrated with PLP and DM20 protein from normal mice. Western analyses showed an altered PLP:DM20 ratio in jpmsd mice relative to wild‐type mice; DM20 levels exceeded PLP levels. It is surprising that a similar pattern of expression was seen in normal mice at <10 days of age: DM20 protein expression preceding PLP expression. Thus, jpmsd mice are capable of synthesizing normal PLP and DM20 protein; however, the PLP gene defect has affected the normal developmental pattern of expression for these two proteins.
Immunological Determination of Galactosylceramide Level in Blood as a Serum Index of Active Demyelination
Authors: Thuillier Y. , Lubetzki C. , Goujet‐Zalc C. , Galli A. , Lhermitte F. , Zalc B.
Published: October 2006

Abstract: An enzyme‐linked immunosorbent assay (ELISA) to determine the level of galactosylceramide (GalC) in biological fluids is described. The assay uses GalC‐coated plastic microtiter plates, with binding of an antibody to GalC detected by a peroxidase‐labeled second antibody. The GalC level was directly estimated in the biological samples, without prior extraction, by competition with the coated hapten. This method allows the detection of 62 pmol of GalC (1.2 nmol/ml). Results using this procedure revealed positive sera only among patients suffering a myelin‐destructive process: either primary, as in multiple sclerosis, or secondary to brain damage, as during ischemic strokes.
Biosynthesis of Angiotensinogen and Angiotensins by Brain Cells in Primary Culture
Authors: Hermann Klaus , Phillips M. Ian , Hilgenfeldt Ulrich , Raizada Mohan K.
Published: October 2006

Abstract: This study focuses on the ability of primary rat brain cells in culture to synthesize angiotensinogen, angio‐tensin I, and angiotensin II. HPLC in combination with radioimmunoassay was used to characterize these compounds. Following incubation with 3H‐labeled isoleucine, radioactively labeled angiotensinogen with an approximate molecular weight of 25,000 was identified in both glial and neuronal cells. Other molecular weight forms of angiotensinogen with molecular weights of about 300 and 160,000 were present in both cell types. In addition to angiotensinogen, radioactively labeled angiotensin I and angiotensin II were also synthesized by neuronal and glial cells. These results suggest that glial and neuronal cells can synthesize angiotensinogen, angiotensin I, and angiotensin II in a similar manner shown for the peripheral renin angiotensin system.
Purification and Characterization of Aromatic L‐Amino Acid Decarboxylase from Rat Kidney and Monoclonal Antibody to the Enzyme
Authors: Shirota Katsumi , Fujisawa Hitoshi
Published: October 2006

Abstract: Aromatic L‐amino acid decarboxylase was purified from rat kidney to homogeneity, as judged by poly‐acrylamide gel electrophoresis, in the presence and absence of sodium dodecyl sulfate (SDS). The final preparation showed an activity of 3,4‐dihydroxyphenylalanine (dopa) decarboxylation of —11,000 nmol/min/mg of protein at 37°C. The purified enzyme also catalyzed the decarboxylation of 5‐hydroxytryptophan, tyrosine, tryptophan, and phenyialanine. The enzyme appeared to be composed of two identical subunits, each possessing a molecular weight of 48,000. The isoelectric point of the enzyme was estimated to be 6.7 in the presence of 8 M urea and 5.60–5.85 in its absence. To examine the identity of aromatic L‐amino acid decarboxylase from various tissues, a monoclonal antibody directed against the enzyme from rat kidney was prepared. Immunotitration and analysis by antibody‐affinity chromatography followed by SDS‐polyacrylamide gel electrophoresis revealed that the enzymes from the stria‐tum, adrenal medulla, pineal gland, liver, and kidney were indistinguishable with respect to immunological cross‐reactivity and molecular size.
Preparation and Characterisation of a Monoclonal Antibody to an Antigen Enriched in Chick Brain Postsynaptic Densities
Authors: Bullock Sarah , Gordon‐Weeks Phillip R. , Csillag Andras
Published: October 2006

Abstract: The preparation and characterisation of a monoclonal antibody to an antigen enriched in day‐old chick brain postsynaptic densities (PSDs), with respect to other subcellular loci, are described. Immunolabelling with this antibody produced a dendritic immunoprecipitate that was markedly stronger in PSDs than in other subcellular loci. Thus, the antiserum could be used as a marker for PSDs during their purification by subcellular fractionation, as well as in the study of PSD assembly. Monoclonal antibody 411B has already been shown to be a useful tool in the chemical determination of changes in synapse density after various experimental manipulations in both the chick and rat. In the present study, we have used the antiserum to monitor the appearance and maintenance or redundancy of synaptic components in the developing chick forebrain.
Autoradiographic Analysis of [3H]Imipramine Binding in the Human Brain Postmortem: Effects of Age and Alcohol
Authors: Gross‐Isseroff Ruth , Biegon Anat
Published: October 2006

Abstract: In vitro quantitative autoradiography of high‐affinity [3H]imipramine binding sites was performed on 16 human brains postmortem. The densities of binding sites were highest in the hypothalamus. Next, in descending order, were the basal and lateral nuclei of the amygdala; substantia innominata; insular cortex; the central nucleus of the amygdala; the anterior nucleus of the thalamus; the head of the caudate nucleus; portions of the frontal, parietal, and temporal cortex; claustrum; the granular layer of the dentate gyrus; substantia nigra; the pyramidal layer of CA fields; globus pallidus; red nucleus; and white matter. Imipramine binding was found to increase with age in a region‐specific manner. The presence of alcohol had a similar effect, which was most pronounced in the hippocampus. Sex and time from death to autopsy did not affect imipramine binding, in our sample.
Inhibition by Cyclic AMP of Phorbol Ester‐Potentiated Norepinephrine Release from Guinea Pig Brain Cortical Synaptosomes
Authors: Shuntoh Hisato , Taniyama Kohtaro , Fukuzaki Hisashi , Tanaka Chikako
Published: October 2006

Abstract: The involvement of Ca2+/pnospholipid‐dependent protein kinase (protein kinase C, PKC) and cyclic AMP‐de‐pendent protein kinase in the K+‐evoked release of norepinephrine (NE) was studied using guinea pig brain cortical synaptosomes preloaded with [3H]NE. 12‐O‐Tetradecanoyl‐phorbol‐13‐acetate (TPA), a potent activator of PKC, enhanced the K+‐evoked release of [3H]NE, in a concentration‐dependent manner, but with no effect on the spontaneous outflow and uptake of [3H]NE in the synaptosomes. The apparent affinity of the evoked release for added calcium but not the maximally evoked release was increased by TPA (10−‐7M). Inhibitors of PKC, polymyxin B, and a more potent inhibitor, staurosporine, counteracted the TPA‐induced po‐tentiation of the evoked release. Both forskolin and dibutyryl cyclic AMP (DBcAMP) enhanced the evoked release, but reduced the TPA‐potentiated NE release. A novel inhibitor of cyclic AMP‐dependent protein kinase, KT5720, blocked both the forskolin‐induced increase in the evoked release and its inhibition of TPA‐induced potentiation in the evoked release, thereby suggesting that forskolin or DBcAMP counteracts the Ca2+‐dependent release of NE by activating cyclic AMP‐dependent protein kinase. These results suggest that the activation of PKC potentiates the evoked release of NE and that the activation of cyclic AMP‐dependent protein kinase acts negatively on the PKC‐activated exocytotic neu‐rotransmitter release process in brain synaptosomes of the guinea pig.
A1 Adenosine Receptor‐G Protein Coupling in Bovine Brain Membranes: Effects of Guanine Nucleotides, Salt, and Solubilization
Authors: Stiles Gary L.
Published: October 2006

Abstract: The effects of guanine nucleotides, NaCl, and solubilization on the interaction of antagonists and agonists with the A1 adenosine receptor of bovine brain membranes were studied using the high‐affinity antagonist radioligand [3H]xanthine amine congener ([3H]XAC). In membranes, guanine nucleotides and NaCl had no effect on [3H]XAC saturation curves. Using agonist (R)‐phenylisopropyladenosine (R‐PIA) competition curves versus [3H]XAC, it was demonstrated that agonists could differentiate two affinity states having high and low affinity for agonist and that guanine nucleotides shifted the equilibrium to an all‐low‐affinity state that was indistinguishable from the low‐affinity state in the absence of guanine nucleotides. In contrast, NaCl decreased agonist affinity by a distinctly different mechanism characterized by a parallel rightward shifted agonist curve such that R‐PIA still recognized two affinity states albeit of lower affinity than in the absence of salt. R‐PIA competition curves in the presence of both guanine nucleotides and salt were still shallow but were shifted far to the right, and two very low affinity states were discerned. On solubilization, guanine nucleotides in a reversible, concentration‐dependent manner increased antagonist ([3H]XAQ but not agonist (R‐N6‐−[3H]phenyl‐isopropyladenosine) binding. This was consequent to a change in maximal binding capacity. R‐PIA competition curves (versus [3H]XAC) in solubilized preparations demonstrated that agonist could still differentiate two agonist specific affinity states which were modulated by guanine nucleotides. In the presence of guanine nucleotides all the receptors were shifted to a uniform low‐affinity state. In contrast, NaCl had no effect on agonist affinity as determined by agonist competition curves in a solubilized receptor preparation. This is firm evidence that guanine nucleotides and NaCl must decrease agonist affinity by distinct mechanisms.
Effects of Increased Extracellular K on the Elemental Composition and Water Content of Neuron and Glial Cells in Leech CNS
Authors: Saubermann Albert J. , Stockton John D.
Published: October 2006

Abstract: Elemental (Na, Cl, K) and water contents of leech (Macrobdella decora) neurons and glial cells were determined under steady‐state exposure to 4, 10, and 20 mM KC1 concentrations (bathing media) using x‐ray microanalysis for quantitative digital imaging of frozen hydrated and dried cryosections. Effects of furosemide, 5‐hydroxytryptamine (5‐HT), and ouabain on elemental distribution changes, induced by exposure to 20 mM K, were also determined. Results demonstrated that packet glial cells and neurons accumulated substantial amounts of K that appeared evenly distributed throughout the cytoplasm. Cell water content also increased as a function of increased cytoplasmic K so that the net effect was an unchanged wet‐weight K concentration (expressed as millimoles per kilogram wet weight). Dry‐weight Na and Cl concentration (expressed as millimoles per kilogram dry weight) increased slightly in glial cells; however, because cell water increased, both Na and Cl (wet‐weight) concentrations decreased. Neurons, in contrast, had no significant change in either Na or K on a wet‐weight basis, so a relatively constant Na/K ratio was maintained despite a small, but significant, increase in K (dry weight) and cell water. These increases, like those in packet glia, were a function of exposure to different concentrations of extracellular space K. These changes were completely abolished by 10−4M ouabain. Neither furosemide nor 5‐HT appeared to affect neuronal or glial K wet‐weight concentrations. These data show that both glial cells and neurons can act as substantial reservoirs for K while maintaining stable K concentrations (by altering cell water content and elemental composition). This process appears to depend on a functioning Na+,K+‐ATPase system.
Astrocyte Metabolism of [15N]Glutamine: Implications for the Glutamine‐Glutamate Cycle
Authors: Yudkoff Marc , Nissim Itzhak , Pleasure David
Published: October 2006

Abstract: The metabolism of glutamine was studied in cultured astrocytes by incubating these cells with [2‐15N]‐glutamine and using gas chromatography‐mass spectrometry to quantitate the transfer of 15N to other amino acids. We found that astrocytes simultaneously synthesize and consume [2‐15N]glutamine, with the respective synthetic and utilization rates being approximately equal (ca. 13.0 nmol min‐1 mg protein‐1). Considerable 15N was transferred to alanine and a significant amount to the essential amino acids leucine, tyrosine, and phenylalanine, the latter process denoting active reamination of cognate ketoacids. A net export of alanine into the medium was noted. Astrocyte glutamine utilization appeared to be mediated via both the phosphate‐activated glutaminase (PAG) pathway and the glutamine aminotransferase pathway, the activity of which was about half that of PAG. The glutamine concentration in the incubation medium determined whether net synthesis or utilization of this amino acid occurred. When glutamine was omitted from the medium, net synthesis occurred. When it was present at a high (5 mM) level, net consumption was observed. At a physiologic (0.5 mM) concentration, neither net synthesis nor consumption was noted, although the 15N data indicated that glutamine was actively metabolized. An implication of this work is that astrocytes clearly are capable of both synthesizing and utilizing glutamine, and current concepts of a glutamate‐glutamine cycle functioning stoichiometrically between astrocytes and neurons may be an oversimplification.
Stimulatory Action of γ‐Aminobutyric Acid on Catecholamine Secretion from Bovine Adrenal Chromaffin Cells Measured by a Real‐Time Monitoring System
Authors: Kataoka Yasufumi , Ohara‐Imaizumi Mica , Ueki Showa , Kumakura Konosuke
Published: October 2006

Abstract: The present study was designed to evaluate the role of γ‐aminobutyric acid (GABA) in the secretory function of cultured chromaffin cells using the method of realtime monitoring. GABA evoked the secretion of catecholamines (CA) from adrenal chromaffin cells in a dose‐dependent manner. Bicuculline 10‐−5M inhibited the stimulatory action of GABA. Diazepam 5 × 10‐−6 and 2.5 × 10‐−5M facilitated the secretory response evoked by 7 × 10‐−5M GABA by 22% and 96%, respectively, which was antagonized by Ro 15–1788. This finding suggests that GABA‐benzodiazepine receptor coupling can function in the secretion of CA from the adrenal chromaffin cells in a manner similar to that observed in the brain. GABA‐evoked release of CA was reduced by 1 μM nifedipine to 16% of control, suggesting the involvement of voltage‐sensitive Ca2+ channels in the mechanisms of the CA‐releasing action of GABA in these cells. From these findings, the involvement of GABAergic mechanisms in the regulation of adrenal medullary function can be proposed.
Imaging of the Degeneration of Neurons and Their Processes in Rat or Cat Brain by 45CaCl2 Autoradiography or 55CoCl2 Positron Emission Tomography
Authors: Gramsbergen J. B. P. , Duin L. Veenma‐v. d. , Loopuijt L. , Paans A. M. J. , Vaalburg W. , Korf J.
Published: October 2006

Abstract: The possibility of using radiolabeled divalent cations to visualize nerve cell degeneration in the brain was investigated after intoxication with neurotoxins. At different survival times after the intracerebral injection of kainic acid or 6‐hydroxydopamine, autoradiographs were made from brain sections of rats that had received 45CaCl2 intravenously 24 h before death. Brain sections, adjacent to those used for autoradiography, of the 6‐hydroxydopa‐mine‐treated rats were used for histofluorescence of catecholamines to check the neurochemical effect of the treatment. These experiments show that radioactive Ca accumulates in brain tissue during a particular phase of degeneration. Not only could degenerating cell bodies be traced by 45Ca autoradiography, but also degenerating nerve terminals in the striato‐nigral and nigro‐striatal projection systems. In positron emission tomography (PET) studies, 55CoCl2 was used as a marker for Ca2+. Unilateral lesions of the cat forebrain, produced by kainic acid, could be imaged in vivo by PET with 55CoCl2. PET with this radiolabel may provide diagnostic potentials for human neurodegenerative disorders.
Relating Cerebral Ischemia and Hypoxia to Insult Intensity
Authors: Emoto Sherrie E. , Kintner Douglas , Feyzi Jan M. , Gilboe David D.
Published: October 2006

Abstract: The contributions of five variables believed to influence the brain's metabolism of O2 during hypoxia [duration, PaO2, ΔCMRO2 (the difference between normal and experimental oxygen uptake), O2 availability (blood O2 content · CBF), and O2 deficit (ΔCMRO2· duration)] were assessed by stepwise and multiple linear regression. Levels of brain tissue carbohydrates (lactate, glucose, and glycogen) and energy metabolites [ATP, AMP, and creatine phosphate (CrP)] were significantly influenced by O2 deficit during hypoxia, as was final CMRO2. After 60 min of reoxygenation, levels of tissue lactate, glucose, ATP, and AMP were related statistically to the O2 deficit during hypoxia; however, CMRO2 changes were always associated more significantly with O2 availability during hypoxia. Creatine (Cr) and CrP levels in the brain following reoxygenation were correlated more to ΔCMRO2 during hypoxia. Changes in some brain carbohydrate (lactate and glucose), energy metabolite (ATP and AMP) levels, and [H+]i induced by complete ischemia were also influenced by O2 deficit. After 60 min of postischemic reoxygenation, brain carbohydrate (lactate, glucose, and glycogen) and energy metabolite (ATP, AMP, CrP, and Cr) correlated with O2 deficit during ischemia. We conclude that “O2 deficit” is an excellent gauge of insult intensity which is related to observed changes in nearly two‐thirds of the brain metabolites we studied during and following hypoxia and ischemia.
Solubilization and Characterization of Substance P Binding Protein from Bovine Brainstem
Authors: Nakata Yoshihiro , Tanaka Hiroyasu , Morishima Yoshiyuki , Segawa Tomio
Published: October 2006

The specific binding protein for substance P (SP) was solubilized in an active form from the crude mitochondrial (P2) fraction of bovine brainstem. After incubation with 3–[(3‐cholamidopropyl)dimethylammonio]‐1‐propane sulfonate (CHAPS) and 0.1 M NaCl at 0°C for 30 min, the SP binding to the supernatant fraction (100,000 g, 60 min) was determined by the glass fiber filtration method reported by Bruns et al. (1983). The specific [3H]SP binding to the solubilized fraction was highly specific for SP and was displaced by nanomolar concentrations of SP and physalaemin, but only by micromolar concentrations of eledoisin. In addition, the binding was inhibited by GTP (approximately 40% of the specific binding decreased by 10 μM GTP) in both preparations. These results were virtually identical to those of P2 membrane preparations and suggested that this high‐affinity SP binding site belongs to the SP‐P type. Scatchard analyses of SP binding to the solubilized fraction revealed a single saturable component with a Bmax of 22.0 ± 5.10 fmol/mg protein and a KD of 0.79 nM, and these values are almost the same as those obtained in the P2 fraction (Bmax= 31.3 ± 3.56 fmol/mg protein, KD= 0.82 nM). Gel filtration analysis showed that the detergent‐SP binding protein complex has two calculated molecular weights of > 1,000,000 and 55,000–60,000 (a corresponding Stokes radius of 35.5 nm).
A 70‐Kilodalton Microtubule‐Associated Protein (MAP2c), Related to MAP2
Authors: Garner Craig C. , Brugg Bernard , Matus Andrew
Published: October 2006

Microtubule‐associated protein 2 (MAP2) from adult brain consists of a pair of high molecular mass (280 kilodaltons) polypeptides, MAP2a and MAP2b. Juvenile brain microtubules also contain a 70‐kilodalton protein that cross‐reacts with monoclonal antibodies against these high molecular weight MAP2s. We have analyzed the relationship between this 70‐kilodalton protein and MAP2 by peptide mapping. Our results show that the 70‐kilodalton species bears strong homology to the MAP2 molecules and that it is distinct from the t MAPs. We propose the name MAP2c for this low molecular weight MAP2 species. MAP2c is developmentally regulated in brain, being more abundant in neonatal tissue than in the adult. In several cell lines, MAP2c is the sole MAP2 species expressed. We examined homogenates from both juvenile brain and MAP2c‐containing cell lines for evidence of a protease activity that might be responsible for generating MAP2c from either MAP2a or MAP2b. No such activity was found, suggesting that MAP2c is an independently synthesized MAP2 species some 200 kilodaltons smaller than the previously recognized forms.
Comparison of 125I‐Angiotensin III and 125I‐Angiotensin II Binding to Rat Brain Membranes
Authors: Abhold Raymond H. , Hanesworth Jodie M. , Harding Joseph W.
Published: October 2006

The binding of 125I‐angiotensin III (125I‐ANG III) to rat brain membranes was examined and compared with that of 125I‐angiotensin II (125I‐ANG II). Degradation of each ligand, as monitored by HPLC, was effectively inhibited using fragments of ANG III and ANG II known to have little affinity for angiotensin binding sites. Three classes of 125I‐ANG III‐binding sites were observed based on affinity (KD= 0.13, 1.83, and 10.16 nM) and capacity (Bmax= 1.30, 18.41, and 67.2 fmol/mg protein, respectively). Two classes of 125I‐ANG II‐binding sites of high affinity (KD= 0.11 and 1.76 nM) and low capacity (Bmax= 1.03 and 18.86 fmol/mg protein, respectively) were also identified. Cross‐displacement studies confirmed that the two highest‐affinity 125I‐ANG Ill‐binding sites and the 125I‐ANG II‐binding sites were the same. On the other hand, the binding of 125I‐ANG III to the low‐affinity 125I‐ANG III‐binding site could not be inhibited with ANG II. These data imply that previously measured differences in the biological potency of cerebroventricularly applied ANG III and ANG II probably do not result from differential binding of these pep‐tides to central angiotensin receptors.
Ontogeny of Dopamine D1 Receptors in Rat Striatum
Authors: Zeng Wanyun , Hyttel John , Murrin L. Charles
Published: October 2006

The development of dopamine D1 receptors in rat striatum during the early postnatal period is described, using [3H]piflutixol as ligand. Dopamine D1 receptors increase in number from day of birth until about 21 days of age, when they reach adult levels. This increase in number parallels the increase in several other dopamine markers in striatum during the same time period. The increase is reflected in an increase in Bmax of ligand binding to D1 receptors. All other properties of D1 receptors that were examined do not change throughout this developmental period and are essentially the same as those found in adult tissue. These include association and dissociation rates, affinity for piflutixol as determined by kinetic and saturation studies, and pharmacology. These studies provide a biochemical and pharmacological basis for further studies on the ontogeny of dopamine receptors and of striatum and on factors regulating development of this region.
Schwann Cell Proliferation Is Accompanied by Enhanced Inositol Phospholipid Metabolism
Authors: Saunders Royal D. , DeVries George H.
Published: October 2006

Inositol phospholipid metabolism during mito‐gen‐induced Schwann cell proliferation has been examined. Addition of axolemma‐ and myelin‐enriched membrane fractions (AXL and MYE, respectively) to cultured Schwann cells stimulated 32P incorporation into phospha‐tidylinositol 4‐monophosphate [Ptdlns(4)P] and phospha‐tidylinositol4,5‐bisphosphate [Ptdlns(4,5)P2]. During the first 5 min of incubation with the mitogens, the amount of 32P incorporated into PtdIns(4)P and PtdIns(4,5)P2 was four‐ to fivefold above control values. The phosphorylation of the inositol phospholipids was dependent on the concentration of membrane mitogens and was maximal within 1 h. Schwann cells that were prelabeled with [3H]glycerol and then stimulated with AXL and MYE displayed a 30–70% increase in the amounts of [3H]PtdIns(4)P and [3H]PtdIns(4,5)P2 and a 60–80% increase in the amount of [3H]phosphatidic acid. A concomitant 20% decrease in the content of [3H]PtdIns was observed after stimulation. These results suggest that the increased metabolism of Ptdlns, Ptdlns(4)P, and Ptdlns(4,5)P2 may be one of the initial molecular events in the transduction of the mito‐genic signal across the Schwann cell plasma membrane.
Sulfhydryl Modification of Two Nicotinic Binding Sites in Mouse Brain
Authors: Stitzel Jerry A. , Campbell Stephen M. , Collins Allan C. , Marks Michael J.
Published: October 2006

Two distinct binding sites with properties corresponding to those expected for nicotinic cholinergic receptors can be identified in brain by the specific binding of nicotine (or acetylcholine) and α‐bungarotoxin. The effects of modification of these binding sites by treatment with the disulfide‐reducing agent dithiothreitol were examined in tissue prepared from DBA mouse brains. Treatment with dithiothreitol reduced the binding measured with either li‐gand, and reoxidization of the disulfides fully restored binding. The effects of dithiothreitol treatment appeared to be due to a reduction in the maximal binding of nicotine and to a decrease in the binding affinity for a‐bungarotoxin. Agonist affinity for the a‐bungarotoxin binding site was reduced bv treatment with low concentrations of dithiothreitol. The nicotine binding sites remaining after disulfide treatment displayed rates of ligand association and dissociation similar to those of unmodified tissue, but treatment of previously unmodified tissue with dithiothreitol accelerated the rate of nicotine dissociation. After reduction, both binding sites could be selectively alkylated with bromoacetyl‐choline. The results suggest that both putative nicotinic receptors in brain respond similarly to disulfide reduction and that their responses resemble those known for the nicotinic receptor of electric tissue.
Neuropeptide‐γ: A Peptide Isolated from Rabbit Intestine that Is Derived from γ‐Preprotachykinin
Authors: Kage R. , McGregor G. P. , Thim L. , Conlon J. M.
Published: October 2006

Abstract: The neurokinin A‐like immunoreactivity in an extract of rabbit small intestine was resolved into two molecular forms by gel permeation chromatography. These components were purified to apparent homogeneity by reverse‐phase HPLC. The primary structure of the larger component was established as the following: Asp‐Ala‐Gly‐His‐Gly‐Gln‐Ile‐Ser‐His‐Lys‐Arg‐His‐Lys‐Thr‐Asp‐Ser‐Phe‐Val‐Gly‐Leu‐Met‐NH2. This amino acid sequence represents residues (72–92) of γ‐preprotachykinin, as predicted from the nucleotide sequence of a cloned cDNA from the rat. The peptide, termed neuropeptide‐γ, lacks residues (3–17) of neuropeptide K, and this segment is specified exactly by exon 4 in the preprotachykinin gene. The smaller form of neurokinin A‐like immunoreactivity was identical to neurokinin A. Neuropeptide K was not present in the extract, demonstrating that the pathways of post‐translational processing of β‐ and γ‐preprotachykinins in the rabbit gut are different.