bluuebunny/crossref_metadata_2025 huggingface dataset:
Dopamine, Serotonin, and Acid Metabolites in Brain Regions from the Developing Offspring of Ethanol‐Treated Rats
Authors: Rathbun Wayne , Druse Mary J.
Published: October 2006

Female rats were pair‐fed control or ethanol liquid diets on a chronic basis prior to parturition. Six brain regions (hypothalamus, cerebellum, brain stem, cortex, corpus striatum, and hippocampus) were dissected from 19‐ and 35‐day‐old rat offspring for the determination of dopamine (DA), sertonin (5‐HT), 3,4‐dihydroxyphenylacetic acid (DOPAC), 5‐hydroxyindole‐acetic acid (5‐HIAA), and homovanillic acid (HVA). DA, 5‐HT, and the acid metabolites were separated simultaneously by reverse‐phase HPLC and were quantitated using electrochemical detection. Between 19 and 35 days of age in control rats we observed an increase in the concentration of 5‐HT and 5‐HIAA in the corpus striatum and hippocampus and a decrease in these compounds in the cerebellum. In addition, there was a development‐related decrease of 5‐HIAA in the hypothalamus and an increase in the brain stem. During the same age period the concentration of dopamine increased in the hypothalamus and corpus striatum. There was also a development‐related decrease in the concentration of DOPAC in the corpus striatum and an increase in the cortex as well as a decrease in HVA in the cerebellum and cortex. In comparison to age‐matched control animals the 19‐ and 35‐day‐old offspring of ethanol‐treated rats had a lower concentration of 5‐HT and/or 5‐HIAA in the cortex, cerebellum, and brainstem. In addition the 35‐day‐old offspring of ethanol‐treated rats exhibited a decrease in DA and HVA in the cortex. The results of the present study suggest that in utero ethanol exposure affects both the serotonergic and dopaminergic systems in brain.
Solubilization and Detergent Effects on Interactions of Some Drugs and Insecticides with the t‐Butylbicyclophosphorothionate Binding Site Within the γ‐Aminobutyric Acid Receptor‐Ionophore Complex
Authors: Seifert Josef , Casida John E.
Published: October 2006

Specific binding of [35S]t‐butylbicyclophosphorothionate (TBPS) to rat brain membranes (RBM) is enhanced nine‐fold by EDTA/water dialysis and 1.3‐ to 4.2‐fold by 50 nM ketosteroid R 5135, or 5 mM 3‐[(3‐cholamidopropyl)dimethylammonio]‐1‐propanesulfonate (CHAPS) or related piperazine‐N‐alkanesulfonate buffers, or extensive washing with NaCl/Na phosphate or Na phosphate/citrate solution. About one‐fifth of the [35S]TBPS binding capacity appears in the soluble fraction whereas the rest remains in particulate form on treatment of the EDTA/water‐dialyzed RBM with 20 mM CHAPS. Similar KD values (64–86 nM) are obtained for the original EDTA/water‐dialyzed membranes and the CHAPS‐treated and/or ‐solubilized preparations. The Bmax of the EDTA‐treated RBM is reduced five‐fold on solubilization with CHAPS. The potency for displacement of [35S]TBPS changes in the presence of CHAPS or on CHAPS solubilization: γ‐aminobutyric acid (GABA) and muscimol inhibit specific [35S]TBPS binding more strongly in the absence than in the presence of CHAPS; TBPS, picrotoxinin, and photoheptachlor epoxide are almost equally active with RBM, RBM + CHAPS, and RBM solubilized with CHAPS. Levels of (1R,αS)‐cis‐cypermethrin and dimethylbutylbarbiturate which are inhibitory with RBM are moderately stimulatory after TBPS receptor solubilization. Thus CHAPS defines three regions of the GABA receptor‐ionophore complex, i.e., the GABA and benzodiazepine receptors, the TBPS/picrotoxinin/polychlorocycloalkane receptor(s), and the sites at which the α‐cyano pyrethroid and the barbiturate interact with TBPS binding.
Hydrolysis of Endogenous Phospholipids by Rat Brain Microsomes
Authors: Witter Brigitte , Kanfer Julian N.
Published: October 2006

Phosphatidylcholine of rat brain microsomes was labeled in vivo by intracerebral injection of either [3H]oleic acid or [methyl‐3H]choline chloride. These labeled microsomes served both as the enzyme source as well as a source of endogenously labeled substrate. Phospholipase D (PLD) activity was detected with these particles only in the presence of exogenous oleate, its activator. Ca2+ and the ionophore A 23187 inhibit PLD activity of oleate‐labeled microsomes. In oleate‐labeled particles, besides phosphatidic acid the product of PLD action radioactivity was also detected in diglyceride as a result of resident phosphatidate phosphohydrolase, which hydrolyzed the phosphatidic acid. The phosphatidate phosphohydrolase could not be completely inhibited by KF and propranolol. The release of endogenous fatty acids from labeled phospholipid by a mellitin‐stimulated phospholipase A2 also present in these particulates produced minimal stimulation of endogenous PLD. Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) are hydrolyzed by 50% in the presence of mellitin and 90% of the radioactivity was found in the lyso‐compounds. Mellitin and oleate together reduced the radioactivity found in lyso‐PC and increased that in lyso‐PE.
Separation of Recycling and Reserve Synaptic Vesicles from Cholinergic Nerve Terminals of the Myenteric Plexus of Guinea Pig Ileum
Authors: Ágoston Dénes V. , Kosh Joseph W. , Lisziewicz Julia , Whittaker Victor P.
Published: October 2006

Acetylcholine‐rich synaptic vesicles were isolated from myenteric plexus‐longitudinal muscle strips derived from the guinea pig ileum by the method of Dowe, Kilbinger, and Whittaker [J. Neurochem.35, 993–1003 (1980)] using either unstimulated preparations or preparations field‐stimulated at 1 Hz for 10 min using pulses of 1 ms duration and 10 V ˙ cm−1 intensity. The organ bath contained either tetradeuterated (d4) choline (50 μM) or [3H]acetate (2 μCi ˙ ml−1); d4 acetylcholine was measured by gas chromatography‐mass spectrometry. As with Torpedo electromotor cholinergic vesicle preparations made under similar conditions the distribution of newly synthesized (d4 or [3H]) acetylcholine in the zonal gradient from stimulated preparations was not identical with that of endogenous (d0, [1H]) acetylcholine, but corresponded to a subpopulation of denser vesicles (equivalent to the VP2 fraction from Torpedo) that had preferentially taken up newly synthesized transmitter. The density difference between the reserve (VP1) and recycling (VP2) vesicles was less than that observed in Torpedo but this smaller difference can be accounted for theoretically by the difference in size between the vesicles of the two tissues. At rest, a lesser incorporation of labelled acetylcholine into the vesicle fraction was observed, and the peaks of endogenous and newly synthesized acetylcholine coincided. Stimulation in the absence of label followed by addition of label did not lead to incorporation of labelled acetylcholine, suggesting that the synthesis and storage of acetylcholine in this preparation and its recovery from stimulation is much more rapid than in Torpedo.
Further Studies on the Nature of Postsynaptic Dopamine Uptake and Metabolism in Rat Striatum: Sodium Dependency and Investigation of a Possible Role for Carrier‐Mediated Uptake into Serotonin Neurons
Authors: Schoepp Darryle D. , Azzaro Albert J.
Published: October 2006

The nature of postsynaptic sites involved in the uptake and metabolism of striatal 3,4‐dihydroxyphenylethylamine (dopamine, DA) was investigated. The accumulation of [3H]DA (10−7M) into slices of rat striatum was found to be greatly dependent (>99%) on the presence of sodium ion in the incubation medium. However, the formation of the [3H]dihydroxyphenylacetic acid (DOPAC) and [3H]homovanillic acid (HVA) was only partially reduced in the absence of sodium (DOPAC, 27% of control; HVA, 47% of control). Inhibition of carrier‐mediated DA neuronal uptake with nomifensine (10−5M) significantly decreased DA accumulation (18% of control) and [3H]DOPAC formation (62% of control), but enhanced [3HVA production (143% of control), Inhibition of the 5‐hydroxytryptamine (5‐HT, serotonin) neuronal uptake system with fluoxetine (10−6M) or selective 5‐HT neuronal lesions with 5,7‐dihydroxytryptamine (5,7‐DHT) had no effect on [3H]DOPAC or [3H]HVA formed from [3H]DA in the presence or absence of nomifensine. These results demonstrate that the uptake and subsequent metabolism of striatal DA to DOPAC and HVA is only partially dependent on carrier‐mediated uptake mechanism(s) requiring sodium ion. These data support our previous findings suggesting a significant role for synaptic glial cell deamination and O‐methylation of striatal DA. Further, experiments with fluoxetine or 5,7‐DHT suggest that 5‐HT neurons do not significantly contribute in the synaptic uptake and metabolism of striatal DA.
Concentration of Free Amino Acids in Primary Cultures of Neurones and Astrocytes
Authors: Patel Ambrish J. , Hunt Anthony
Published: October 2006

The cellular distribution of free amino acids was estimated in primary cultures (14 days in vitro) composed principally of cerebellar interneurones or cerebellar and forebrain astrocytes. In cultured neural cells, the overall concentration of amino acids resembled that found in brain at the corresponding age in vivo. In the two neural cell types, there were marked differences in the distribution of amino acids, in particular, those associated with the metabolic compartmentation of glutamate. In neuronal cell cultures, the concentrations of glutamate, aspartate, and γ‐aminobutyric acid were, respectively, about three, four, and seven times greater than in astrocytes. By contrast, the amount of glutamine was $65% greater in astroglial cell cultures than in interneurone cultures. An unexpected finding was a very high concentration of glycine in astrocytes derived from 8‐day‐old cerebellum, but the concentrations of both serine and glycine were greater in nerve cell cultures than in forebrain astrocytes. The essential amino acids threonine, valine, isoleucine, leucine, tyrosine, phenylalanine, histidine, lysine, and arginine were all present in the growth medium, and small cellular changes in the contents of some of these amino acids may relate to differences in their influx and efflux during culturing and washing procedures. The present results, together with our previous findings, provide further support for the model assigning the “small” compartment of glutamate to glial cells and the “large” compartment to neurones, and also underline the metabolic interaction between these two cell types in the brain.
Expression of Hyaluronectin by C‐6 Glial Cells at High Density
Authors: Bhat Narayan R. , Brunngraber Eric G. , Delpech Bertrand
Published: October 2006

Hyaluronectin, a brain glycoprotein that has been localized to the nodes of Ranvier in vivo and to oligodendrocytes in primary cultures of neonatal rat brain cells, was shown by using an unlabeled immunoperoxidase method to be present in C‐6 glial cells grown to high density. The density‐dependent expression of this glycoprotein is in accordance with the known properties of the glial stem cells, i.e., induction of differentiated properties such as 2′,3′‐cyclic nucleotide‐3′‐phosphohydrolase, glutamine synthetase, S‐100 protein, and glial fibrillary acidic protein.
A Biochemical Explanation of Phenyl Acetate Neurotoxicity in Experimental Phenylketonuria
Authors: Loo Yen Hoong , Potempska Anna , Wisniewski Henry M.
Published: October 2006

Abstract: The in vivo formation of [1‐14C]acetyl‐coenzyme A from D‐[3‐14C]3‐hydroxybutyrate in the brain of the suckling rat was not affected by postnatal exposure to phenyl acetate. However, utilization of the generated acetyl‐coenzyme A was significantly inhibited in certain metabolic reactions, namely synthesis of fatty acids and of sterols, but not in others as the Krebs cycle reactions that lead to the production of dicarboxylic amino acids. The incorporation of D‐[U‐14C]glucosamine into N‐acetylneuraminic acid bound to glycoproteins was appreciably diminished in the rat pup previously exposed to maternal phenylketonuria induced by phenyl acetate. During the period of very rapid development of the brain, interference by phenyl acetate and/or its metabolites with certain critical biosynthetic pathways that require acetylcoenzyme A would significantly contribute to retarded maturation of the brain that occurs in phenylketonuria.
Effects of Aging and Morphine Administration on Calmodulin and Calmodulin‐Regulated Enzymes in Striata of Mice
Authors: Hoskins B. , Ho I. K. , Meydrech E. F.
Published: October 2006

Abstract: Male ICR mice, young (25‐days old), mature (3‐months old), and old (22 months), were injected with morphine sulfate (10 mg/kg, s.c.) or were implanted with morphine pellets (75 mg). Controls received saline injections or placebo pellets. One hour after injections and 72 h after pellet implantations, the mice were decapitated and striatal regions were removed for the following analyses: calmodulin (CaM) levels via radioimmunoassay and activities of cyclic nucleotide phosphodiesterases, adenylate and guanylate cyclases, and Ca2+, Mg2+‐ATPase. Acute morphine treatment produced the following: (1) increases in calmodulin levels in the young and old mice while having no effect on mature levels; (2) increases in activities of guanylate cyclase of mature mice while decreasing those of the old mice; (3) no effects on activity of adenylate cyclase; (4) decreased activity of cyclic AMP‐phosphodiesterase in young mice only; (5) decreased activity of Ca2+, Mg2+‐ATPase in the old mice only. The only changes found in striata from morphinetolerant mice when compared with age‐matched controls were elevations in cyclic GMP‐phosphodiesterase activities in all three age groups. Differences in control values of the three age groups were as follows: CaM levels, mature > old > young; Ca2+, Mg2+‐ATPase activity, old > mature‐young. The results indicate age‐induced changes in cellular regulation and biochemical responses to morphine.
Characterization of Neutral and Acidic Glycosphingolipids in Brains of Two Patients with GM1 Gangliosidosis Type 1 and Type 2
Authors: Ishikawa Yukitoshi , Makita Akira , Minami Ryoji
Published: October 2006

Abstract: Brains of two patients with GM1 gangliosidosis type 1 and type 2, together with the age‐matched control brains, were analyzed for glycosphingolipids. Six species of neutral glycolipids, eight species of gangliosides, and sulfatide were isolated from the diseased brains and identified. In addition to GM1 ganglioside and its asialo derivative, the diseased brains accumulated considerable amounts of gangliotriaosylceramide and glycolipids belonging to the globe series, the accumulation of which cannot be explained by deficient β‐galactosidase activity in this disease. GM4 ganglioside was detected in the type 2 brain, but not in type 1. As to fatty acid composition of monohexosylceramides and sulfatide in the two diseased brains, stearic acid was more predominant in the type 1 brain than in the type 2 brain. In light of our previous observations on a Tay‐Sachs brain and present results, it appears that metabolism of the globo series glycolipids, which is active in normal brain at early infancy but inactive thereafter, remains in brains with GM1 gangliosidosis (types 1 and 2) and Tay‐Sachs disease, reflecting a disturbance in development of the brain.
Large‐Scale Purification of Torpedo Electric Organ Synaptosomes
Authors: Israël M. , Lazereg S. , Lesbats B. , Manaranche R. , Morel N.
Published: October 2006

Abstract: A procedure for the large‐scale purification of Torpedo electric organ synaptosomes is described. The synaptosomal fraction obtained is very pure as judged from biochemical and morphological data. In addition, acetylcholine (ACh) release was demonstrated after KCl depolarization of synaptosomes in the presence of calcium. Two hundred grams of electric organ can be fractionated in a single run, allowing biochemical studies on presynaptic membrane constituents.
Cystathionine Accumulation in Various Regions of Brain of DL‐Propargylglycine‐Treated Rats
Authors: Kodama Hiroyuki , Ikeda Hisao , Awata Shiro , Nakayama Kazuko
Published: October 2006

Abstract: The contents of cystathionine and taurine, as well as cystathionine β‐synthase activity in various regions of the brains of normal and DL‐propargylglycinetreated rats, were measured. The content of cystathionine in each region of brain increased gradually from 0.5 mg to 20 mg/200 g body weight in relation to the dose of DL‐propargylglycine. Cystathionine was found to be unevenly distributed in brains of both normal and DL‐propargylglycine‐treated rats. On the other hand, the activity of cystathionine β‐synthase was evenly distributed in various regions of normal rat brain, and was unaltered following treatment of rats with DL‐propargylglycine. The concentration of taurine was similarly unaffected by DL‐propargylglycine injection.
Low Activity of Angiotensin‐Converting Enzyme in Cerebral Microvessels of Young Spontaneously Hypertensive Rats
Authors: Kobayashi Hideyuki , Wada Akihiko , Izumi Futoshi , Take Keiko , Magnoni Maria Sandra
Published: October 2006

Abstract: Angiotensin‐converting enzyme (ACE) activity was measured in microvessels prepared from cerebral cortices of 4‐week‐old spontaneously hypertensive rats (SHR). The Vmax value of the ACE activity in the cerebral microvessels of SHR was lower than that of Wistar Kyoto controls of the same age by 25% without difference in Km value for substrate. The low activity of ACE in the cerebral microvessels of young SHR indicates that in this animal model of hypertension the function of ACE is genetically altered in the cerebral microvessels, which may be correlated with the alteration of the cerebral microcirculation and pathogenesis of hypertension.
Role of Hyperthermia in Effects of Electroconvulsive Shock on Protein Synthesis in the Rabbit
Authors: Nowak Thaddeus S.
Published: October 2006

Abstract: The effects of electroconvulsive shock (ECS) on rectal temperature (TR) and on protein synthesis in brain and liver were compared in rabbit, rat, and mouse. Protein synthesis status was assessed using an in vitro amino acid incorporation method which provides information equivalent to polyribosome profiles. In the rabbit, TR rose from 39.5 ± 0.4°C to 40.4 ± 0.2°C within 10 min following a single ECS, and significant hyperthermia persisted for at least 60 min. This effect was markedly attenuated in animals housed at 4°C. In vitro protein synthesis activities of rabbit brain and liver preparations were significantly reduced following ECS only in those animals whose TR exceeded 40°C. In the rat, ECS gave rise to a significant hyperthermia, but in no case did TR exceed 40°C, and protein synthesis activity of brain supernatants was not affected. In the mouse, ECS reduced TR and had no effect on in vitro protein synthesis activity. These results demonstrate that the unique sensitivity of protein synthesis in rabbit tissues to electroconvulsive shock is a direct consequence of the hyperthermia that arises following ECS in this species.
Calcium‐Dependent Transglutaminase of Rat Sympathetic Ganglion in Development and After Nerve Injury
Authors: Gilad Gad M. , Varon Larry E. , Gilad Varda H.
Published: October 2006

Abstract: The activity of transglutaminase (TG) was examined in the rat superior cervical ganglion (SCG) during development and after postganglionic nerve crush. During postnatal development the enzyme activity is increased by sevenfold in parallel to protein content of the ganglion and reaches adult levels by day 35 after birth. The endogenous activity (enzyme activity assayed in the absence of the exogenous substrate) during development is transiently elevated with a peak at day 21 postnatal. In the adult ganglion the enzyme specific activity is evenly distributed in all subcellular compartments, but most of it is contained in the cytosol. Within the first hour after axotomy TG activity is rapidly and transiently elevated. The peak value, 80% above control levels, is attained by 30 min postoperative. At this time the activity is increased in all subcellular fractions, but the endogenous activity is selectively increased in the fraction containing nuclei. The enhanced TG activity after axotomy can be prevented by topical treatments with verapamil, an inhibitor of voltage‐dependent calcium fluxes across excitable membranes, or with the calcium chelator EGTA. The results show that intracellular TG activity is present in the SCG and that it increases with postnatal growth of the ganglion. After axotomy the enzyme activity is rapidly and transiently increased in the ganglion and this elevation critically depends on calcium fluxes.
Identification of Endogenous Calmodulin‐Dependent Kinase and Calmodulin‐Binding Proteins in Cold‐Stable Microtubule Preparations from Rat Brain
Authors: Larson Roy E. , Goldenring James R. , Vallano Mary Lou , DeLorenzo Robert J.
Published: October 2006

Abstract: Calmodulin‐dependent kinase activity was investigated in cold‐stable microtubule fractions. Calmodulin‐dependent kinase activity was enriched approximately 20‐fold over cytosol in cold‐stable microtubule preparations. Calmodulin‐dependent kinase activity in cold‐stable microtubule preparations phosphorylated microtubule‐associated protein‐2, α‐ and β‐tubulin, an 80,000‐dalton doublet, and several minor phosphoproteins. The endogenous calmodulin‐dependent kinase in cold‐stable microtubule fractions was identical to a previously purified calmodulin‐dependent kinase from rat brain by several criteria including (1) subunit molecular weights, (2) subunit isoelectric points, (3) calmodulin‐binding properties, (4) subunit autophosphorylation, (5) calmodulin‐binding subunit composition on high‐resolution sodium dodecyl sulfate‐polyacrylamide gel electrophoresis, (6) isolation of kinase on calmodulin affinity resin, (7) kinetic parameters, (8) phosphoamino acid phosphorylation sites on β‐tubulin, and (9) phosphopeptide mapping. Endogenous cold‐stable calmodulin‐dependent kinase activity was isolated from the microtubule fraction by calmodulin affinity resin column chromatography and specifically eluted with EGTA. This kinase fraction contained the calmodulin‐binding, autophosphorylating p and σ subunits of the previously purified kinase. The p and σ subunits of this kinase represented the major calmodulin‐binding proteins in the cold‐stable microtubule fractions as assessed by denaturing and nondenaturing procedures. These results indicate that calmodulin‐dependent kinase is a major calmodulin‐binding enzyme system in cold‐stable microtubule fractions and may play an important role in mediating some of the effects of calcium on microtubule and cytoskeletal dynamics.
Nerve Growth Factor Affects Cyclic AMP Metabolism, but Not by Directly Stimulating Adenylate Cyclase Activity
Authors: Race Holly M. , Wagner John A.
Published: October 2006

Abstract: Published experiments both support and contradict the hypothesis that nerve growth factor (NGF) can regulate adenylate cyclase activity. Using a sensitive assay that measures the conversion of [2‐3H]adenine to [3H]cyclic AMP, we have shown that NGF alone cannot measurably stimulate cyclic AMP production, whereas the adenosine analog phenylisopropyladenosine (PIA) stimulates adenylate cyclase 20‐fold over basal activity. NGF potentiates the capacity of both PIA and cholera toxin to stimulate cyclic AMP accumulation at all concentrations tested. This potentiation occurs at the earliest measurable times and does not require RNA synthesis. Therefore, we conclude that cyclase activation alone does not account for the effect of NGF on cyclic AMP accumulation and we discuss possible mechanisms.
Effect of Hyperglycemia and Its Prevention by Insulin Treatment on the Incorporation of 32P into Polyphosphoinositides and Other Phospholipids in Peripheral Nerve of the Streptozotocin Diabetic Rat
Authors: Berti‐Mattera Liliana , Peterson Richard , Bell Margaret , Eichberg Joseph
Published: October 2006

Abstract: The influence of varying doses of streptozotocin and preventive insulin treatment on phospholipid metabolism in sciatic nerve in vitro from diabetic rats was studied. Animals were given 30, 45, and 60 mg/kg injections of streptozotocin and 10 weeks later nerves were removed and incubated in the presence of [32P]orthophosphate. The quantity of isotope incorporated into phosphatidylinositol‐4,5‐bisphosphate (PIP2) was progressively greater with increasing drug dosage, whereas uptake of label into other phospholipids was unchanged. Rats were made diabetic and within 72 h were implanted with long‐acting, insulin‐containing osmotic minipumps and the incorporation of [32P]orthophosphate into phospholipids of intact and epineurium‐free nerves was examined 8 weeks later. For whole nerve, increased labeling in nerves from diabetic animals occurred only in PIP2 and phosphatidylinositol‐4‐phosphate (PIP) and was completely prevented by insulin treatment. Isotope incorporation into polyphosphoinositides was also markedly elevated (100%) in desheathed diabetic nerves, but not in nerves from insulin‐treated animals. Other phospholipids in epineurium‐free nerves displayed some rise in isotope uptake, but the increases were not prevented by insulin treatment and appeared unrelated to hyperglycemia. Morphological examination of nerves extended previous findings that prolonged insulin treatment produces axonal degeneration. These observations indicate that abnormal nerve polyphosphoinositide metabolism is at least in part a consequence of hyperglycemia. The metabolic alterations may be intimately involved in reduced nerve conduction velocity, which is characteristic of diabetic neuropathy.
Dynamic Aspects of Glucocorticoid Receptors in the Spinal Cord of the Rat
Authors: Ortí Eduardo , Tornello Silvia , De Nicola Alejandro F.
Published: October 2006

Abstract: In spite of biochemical and autoradiographic evidence for glucocorticoid binding sites in the spinal cord (SC), events occurring after the preliminary step of hormone binding were not studied. In this investigation, we have examined the transformation (activation) of the cytosolic receptor coupled to [3H]dexamethasone (DEX) and the in vivo interaction of adrenal hormone [corticosterone (CORT)] with purified nuclei from the SC, in addition to the CORT content of the SC before and after stress. Binding of [3H]DEX in the SC was 40% lower than in the hippocampus (HC), although the KD values were comparable. Transformation of [3H]DEX‐receptor complexes in the cytosol was demonstrated by diethylaminoethane‐cellulose chromatography, by DNA‐cellulose binding, and by a combined minicolumn procedure including hydroxyapatite in addition to the last two techniques for separation of transformed, nontransformed, and meroreceptor complexes. In all these situations, SC glucocorticoid binding sites behaved similarly to those in the HC. Nuclear uptake of a tracer dose of [3H]CORT was much lower in the SC than in the HC; nuclear retention of CORT was more easily detected by radioimmunoassay after injection of 1 mg of CORT into adrenalectomized rats. Substantial amounts of CORT, which increased in level after stress, were measured in five regions in the SC, with higher concentrations in the cervical regions. These studies suggest that although SC and HC receptors show similar properties in vitro, differences emerged at the level of nuclear uptake in vivo, in that glucocorticoid action in the SC was similar to that in the optic nerve, where receptors seem to be localized mostly in glial cells.
Normal Myelin Composition and Phospholipid Synthesis in Adrenalectomized Rats with Reduced Brain Myelin and Glycerol 3‐Phosphate Dehydrogenase Activity
Authors: Preston Sheryl L. , McMorris F. Arthur
Published: October 2006

Abstract: The composition of CNS myelin was investigated in rats adrenalectomized at day 14 and killed 7 days later, previously shown to result in a 25% reduction in the amount of bulk‐isolated myelin and a 40% decrease in brain glycerol 3‐phosphate dehydrogenase activity. The proportions of the major myelin proteins, as well as the specific activity of 2′,3′‐cyclic nucleotide 3′‐phosphohydrolase, were the same in the myelin from both adrenalectomized and control animals. The amount of total phospholipid and the proportions of individual phospholipids were also normal in myelin from the adrenalectomized animals. The amount of nonmyelin phospholipid in whole brain was unchanged by adrenalectomy. Labeling studies carried out 4 days after adrenalectomy of 14‐day‐old animals showed no change in the synthesis rates of the major myelin phospholipids as compared with the synthesis rate of nonmyelin phospholipids. Furthermore, incorporation of [1,(3)‐14C]glycerol into the glycerol moiety of ethanolamine plasmalogen, which requires glycerol 3‐phosphate dehydrogenase, was also normal, showing that the reduced oligodendroglial glycerol 3‐phosphate dehydrogenase activity following adrenalectomy was not rate‐limiting for myelin phospholipid synthesis.
Incorporation of Acetate into Acetylcholine, Acetylcarnitine, and Amino Acids in the Torpedo Electric Organ
Authors: Corthay Jacqueline , Dunant Yves , Eder Lorenza , Loctin Françoise
Published: October 2006

Abstract: The metabolism of acetate was investigated in the nerve‐electroplaque system of Torpedo marmorata. In intact fragments of electric organ, radiolabeled acetate was incorporated into acetylcholine (ACh), acetylcarnitine (ACar), and three amino acids: aspartate, glutamate, and glutamine. These compounds were identified by TLC, high‐voltage electrophoresis, column chromatography, and enzymic tests. The system responsible for acetate transport and incorporation into ACh displayed a higher affinity but a lower Vmax than that involved in the synthesis of ACar and amino acids. Choline, when added to the medium, increased the rate of acetate incorporation into ACh but decreased (at concentrations >10−5M) that into ACar and amino acids. Monofluoroacetate slightly depressed ACh and ACar synthesis from external acetate but inhibited much more the synthesis of amino acids. During repetitive nerve stimulation, the level of the newly synthetized [14C]ACh was found to oscillate together with that of endogenous ACh, but the level of neither [14C]ACar nor the 14C‐labeled amino acids exhibited any significant change as a function of time. This means that there is probably no periodic transfer of acetyl groups between ACh and the investigated metabolites in the course of activity. Acetate metabolism was also tested in the electric lobe (which contains the cell bodies of the neurons innervating the electric organ) and in Torpedo synaptosomes (which are nerve terminals isolated from the same neurons). Radioactive pyruvate and glutamine were also assayed in some experiments for comparison with acetate. These observations are discussed in connection with ACh metabolism under resting and active conditions in tissues where acetate is the preferred precursor of the neurotransmitter.
Demonstration of an Autoreceptor Modulating the Release of [3H]5‐Hydroxytryptamine from a Synaptosomal‐Rich Spinal Cord Tissue Preparation
Authors: Monroe Philip J. , Smith David J.
Published: October 2006

Abstract: A superfusion system employed to measure the K+ ‐stimulated release of [3H]5‐hydroxytryptamine ([3H]5‐HT,[3H]serotonin) from a synaptosomal‐rich spinal cord tissue preparation was carefully characterized, then used to examine the regulation of spinal 5‐HT release. Spinal 5‐HT release is apparently modulated by an autoreceptor. Exogenous 5‐HT depressed, in a concentration‐dependent manner, the K+‐stimulated release of [3H]5‐HT. Similarly, lysergic acid diethylamide (LSD) produced a concentration‐dependent decrease in [3H]5‐HT release. Methiothepin and quipazine blocked the inhibition of release induced by exogenous 5‐HT. The 5‐HT2 receptor antagonists spiperone and ketanserin failed to alter the action of 5‐HT at the spinal 5‐HT autoreceptor. Spiperone and ketanserin were shown, however, to alter the storage of [3H]5‐HT. When used in concentrations greater than 10 nM, the drugs evoked increases in basal [3H]5‐HT and [3H]5‐hydroxyindoleacetic acid ([3H]5‐HIAA) effluxes which were independent of the presence of calcium ions. A good agreement existed between the potencies of drugs for modifying autoreceptor function and their abilities to compete for high‐affinity [3H]5‐HT binding in the spinal cord (designated 5‐HT1). Furthermore quipazine, in concentrations that preferentially interact with the 5‐HT1B subtype, antagonized the actions of exogenous 5‐HT on K+ ‐stimulated release. Spiperone, in a concentration that approximated the affinity constant of 5‐HT1A sites for the drug, was ineffective in altering the ability of exogenous 5‐HT to modulate K+‐stimulated [3H]5‐HT release. These results suggest that 5‐HT1B sites are associated with serotonergic autoreceptor function in the spinal cord.
Effects of Substance P on Carbachol‐Stimulated 45Ca2+ Uptake into Cultured Adrenal Chromaffin Cells
Authors: Boksa P.
Published: October 2006

Abstract: Substance P is known to modulate acetylcholine‐induced catecholamine release from adrenal chromaffin cells. To investigate the mechanisms involved in this modulation, the present study examined the effects of substance P on net 45Ca2+ fluxes in cultures of bovine adrenal chromaffin cells. Two effects of substance P were observed: (1) Substance P inhibited carbachol‐induced 45Ca2+ uptake and 45Ca2+ efflux and (2) substance P protected against desensitization of carbachol‐induced 45Ca2+ uptake and 45Ca2+ efflux. Thus substance P modulates two other cholinergic responses, 45Ca2+ uptake and 45Ca2+ efflux, in a manner similar to its modulation of catecholamine release. The results also indicate that substance P's inhibition of net carbachol‐induced 45Ca2+ uptake is due to inhibition of 45Ca2+ uptake rather than enhancement of 45Ca2+ efflux. Substance P almost completely inhibited carbachol‐induced 45Ca2+ uptake in both Na+‐containing and Na+‐free media, suggesting that substance P can inhibit the uptake of 45Ca2+ induced by carbachol regardless of whether 45Ca2+ is taken up through voltage‐sensitive or acetylcholine receptor‐linked channels. However, substance P produced only a small inhibition of K+‐induced 45Ca2+ uptake, indicating that substance P does not interact directly with voltage‐sensitive Ca2+ channels. In addition, substance P's inhibition of carbachol‐induced 45Ca2+ uptake was noncompetitive with respect to Ca2+, and increasing concentrations of extracellular Ca2+ were unable to overcome substance P's inhibition of [3H]‐norepinephrine ([3H]NE) release. It is concluded that substance P does not interact directly with Ca2+ channels in bovine adrenal chromaffin cells.
Maitotoxin‐Induced Release of γ‐[3H]Aminobutyric Acid from Cultures of Striatal Neurons
Authors: Shalaby Ismail A. , Kongsamut Sathapana , Miller Richard J.
Published: October 2006

Abstract: The potent marine toxin, maitotoxin, induced the release of γ‐[3H]aminobutyric acid (GABA) from reaggregate cultures of striatal neurons in a dose‐dependent manner. Maitotoxin‐induced release occurred following a lag period of several minutes and was persistent. Release induced by 70 mM K+ on the other hand was immediate and transient in nature. Co2+ (3 mM) and Cd2+ (1 mM) inhibited maitotoxin‐induced release of GABA as did removal of extracellular Ca2+. However, the organic calcium antagonists nisoldipine, nitrendipine, and D‐600 at concentrations of 10−6M did not block maitotoxin‐induced or 70 mM K+‐induced release. High concentrations of D‐600 (10−4M) partially blocked both maitotoxin‐ and 70 mM K+‐induced release. The dihydropyridine calcium agonist BAY K8644 (10−6M) did not enhance maitotoxin‐induced or 70 mM K+‐induced release. Replacement of Na+ in the incubation medium with choline led to an increased basal output of GABA and an apparent inhibition of the effect of maitotoxin. These data are discussed with reference to the hypothesis that maitotoxin can directly activate voltage‐sensitive calcium channels.
Effect of 2‐(4‐Phenylpiperidino)cyclohexanol on Acetylcholine Release and Subcellular Distribution in Rat Striatal Slices
Authors: Říčný J. , Collier B.
Published: October 2006

Abstract These experiments measured the effect of 2‐(4‐phenylpiperidino)cyclohexanol (AH5183) on the release of acetylcholine (ACh) and its subcellular distribution in slices of rat striatum incubated in vitro. The AH5183, a drug that blocks the uptake of ACh by isolated synaptic vesicles, reduced the release of ACh from slices stimulated to release transmitter in response to K+ depolarization. Tissue stimulated in the presence of AH5183 contained more ACh in a nerve terminal cytoplasmic fraction than did tissue stimulated in the drug's absence, but stimulation in AH5183's presence reduced the amount of ACh measured in fractions containing synaptic vesicles. The depletion of ACh caused by stimulating tissue in the presence of AH5183 was more evident in the fraction of nerve terminal ACh occluded within synaptic vesicles as isolated by gradient centrifuga‐tion (fraction D) than it was in other nerve terminal occluded stores. It is concluded that the synaptic vesicles isolated as fraction D under the present experimental conditions likely contain releasable transmitter. The AH5183 also depressed the spontaneous release of ACh from incubated slices of striatum and this effect was evident in the presence or the absence of medium Ca2+. It is suggested that this effect might indicate that the process of spontaneous ACh release measured neurochemically results, in part, from an AH5183‐sensitive carrier‐mediated process.
A Radioimmunoassay for the Phosphoprotein B‐50: Distribution in Rat Brain
Authors: Oestreicher A. B. , Dekker L. V. , Gispen W. H.
Published: October 2006

Abstract: A radioimmunoassay (RIA) for the B‐50 protein was developed to determine B‐50 in total homogenates of rat tissues. A tracer of purified B‐50 was prepared at high activity (10–30 μCi/μg protein) by phosphorylating B‐50 with carrier‐free [γ‐32P]ATP, catalyzed by purified protein kinase C. The RIA was performed using affinity‐purified anti‐B‐50 immunoglobulins G in a detergent containing medium and detected B‐50 at levels of 0.1–10 ng. Specificity of the antibodies was ascertained by immunoprecipitation of B‐50 from a crude mitochondrial membrane fraction from rat brain and by immunoblotting. For the B‐50 content in rat brain the following distribution pattern was found: medulla spinalis < cerebellum < hippocampus; cerebral cortex < periaqueductal gray < septum. The septum contained 80 μg/g tissue weight. The level in liver homogenates was below detection. The regional distribution is in fair agreement with the pattern of the endogenous B‐50 phosphorylation in rat brain synaptosomal plasma membranes previously reported.
Separation of Assembly‐Competent Tubulin from Brain Microtubule Protein Preparations Using a Fast‐Performance Liquid Chromatography Procedure
Authors: Roychowdhury Sukla , Gaskin Felicia
Published: October 2006

Abstract: Fast‐performance liquid chromatography was used to purify assembly‐competent tubulin from porcine brain microtubule protein prepared by two cycles of assembly‐disassembly. Microtubule protein (1–100 mg at 1.5–2.5 mg/ml) in buffer consisting of 0.1 M 2‐(N‐morpholino)ethanesulfonic acid, 0.5 mM MgCl2, 1 mM EGTA, 0.3 M KCl, and 0.02 mM GTP (pH 6.6) was applied to the Mono Q column (anion exchanger). The microtubule‐associated proteins, GTP and GDP, eluted in the void volume. The tubulin fraction eluted at 0.45–0.50 M KCl with 65–80% recovery. The tubulin fraction contained trace enzymatic activities when compared with the starting microtubule protein, i.e., < 1 versus 60 mU/mg/min of nucleotide diphosphate kinase, 0.2 versus 7.0 nmol/mg/min of Mg‐ATPase at pH 6.6, and 0.2 versus 88 mU/mg/min of adenylate kinase. Both the Mono Q‐purified tubulin and the pelleted microtubules that were assembled in 0.5 mM [3H]GTP contained 0.77 mol of labeled nucleotide/tubulin dimer. The Mono Q‐purified tubulin fraction was competent to assemble, i.e., the critical concentration was 0.1 mg/ml in the presence of 0.03 mM taxol and 1 mM GTP at 37°C. The Mono Q‐purified tubulin fraction showed trace high‐molecular‐weight components, which were removed on Mono S (cation exchanger) columns. Alternatively, microtubule protein in buffer was applied to the Mono S column. Tubulin, trace nontubulin proteins, and several enzymatic activities came off in the void volume. A combination of Mono Q‐Mono S or Mono S‐Mono Q chromatography resulted in highly purified protein. However, it is possible to separate active tubulin‐nucleotide rapidly from the major microtubule‐associated proteins, enzyme contaminants, and unbound nucleotide with just Mono Q column chromatography.
Studies on the Neurotoxicity of 1‐Methyl‐4‐Phenyl‐1,2,3,6‐Tetrahydropyridine: Inhibition of NAD‐Linked Substrate Oxidation by Its Metabolite, 1‐Methyl‐4‐Phenylpyridinium
Authors: Vyas Ivy , Heikkila Richard E. , Nicklas William J.
Published: October 2006

Abstract: The effects of the parkinsonism‐inducing neurotoxin 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) and its 4‐electron oxidation product 1‐methyl‐4‐phenylpyridinium (MPP+) were studied in isolated mitochondria and in mouse brain striatal slices. ADP‐stimulated oxidation of NAD‐linked substrates was inhibited in a time‐dependent manner by MPP+ (0.1–0.5 mM), but not MPTP, in mitochondria prepared from rat brain, mouse brain, or rat liver. Under identical conditions, succinate oxidation was relatively unaffected. In neostriatal slices prepared from the mouse, a species susceptible to the dopaminergic neurotoxicity of MPTP, incubation with either MPP+ or MPTP caused metabolic changes consistent with inhibition of mitochondnial oxidation, i.e., an increase in the formation of lactate and accumulation of the amino acids glutamate and alanine with concomitant decreases in glutamine and aspartate levels. The changes resulting from incubation with MPTP were prevented by the monoamine oxidase inhibitor pargyline, which blocks formation of MPP+ from MPTP. The results suggest that compromise of mitochondrial function and its metabolic sequelae within dopaminergic neurons could be an important factor in the neurotoxicity observed after MPTP administration.
Atrial Natriuretic Hormones Raise the Level of Cyclic GMP in Neural Cell Lines
Authors: Friedl Arno , Harmening Christine , Hamprecht Bernd
Published: October 2006

Abstract: Atriopeptin III and related atrial natriuretic peptide hormones strongly elevate the level of cyclic GMP in three neural tumor cell lines. At peptide concentrations of 1 γM clear‐cut plateaus of the dose‐response curves are not yet reached. Atriopeptin III increases the intracellular concentration of cyclic GMP to a maximum in the course of 30–40 min. The effect of atriopeptin III on the cellular cyclic GMP level is independent of the concentration of extracellular Ca2+ and is not affected by the Ca2+ ionophore A23187. These results suggest (1) that atrial natriuretic hormones may play an important role in the nervous system, and (2) that cultured neural cells may be useful tools in the elucidation of the mechanisms of action of these hormones.
Interaction of t‐Butylbicyclophosphorothionate with γ‐Aminobutyric Acid‐Gated Chloride Channels in Cultured Cerebral Neurons
Authors: Tehrani Mohammad H. Jalilian , Vaidyanathaswamy Ramamurthy , Verkade John G. , Barnes Eugene M.
Published: October 2006

Abstract: The role of t‐butylbicyclophosphorothionate (TBPS) as an antagonist of γ‐aminobutyric acid (GABA) was studied with primary cultures of neurons from the chick embryo cerebrum. The addition of GABA stimulated the uptake of 36Cl− by neurons and the dose dependence of this effect followed hyperbolic kinetics with a K0.5= 1.3 μM for GABA. TBPS proved to be a potent inhibitor of GABA‐dependent Cl− uptake (IC50= 0.30 μM). Analysis of the kinetics of this process revealed that TBPS is a noncompetitive inhibitor (Ki= 0.15 μM) with respect to GABA. Scatchard analysis of direct binding of [35S]TBPS to membranes isolated from neuronal cultures gave curvilinear plots. These could be resolved by nonlinear regression methods into two components with KD values of 3.1 nM and 270 nM. The TBPS binding constant for this lower affinity site agreed well with the IC50 and Ki values for inhibition of Cl− flux, suggesting that this site is physiologically relevant to GABA antagonism. GABA was a noncompetitive displacer of [35S]TBPS binding to the lower affinity site. The Ki value for this displacement by GABA (1.7 μM) was comparable to the value for GABA enhancement of Cl− flux. The binding of [35S]TBPS to its low‐affinity site on neuronal membranes was ninefold higher in the presence of Cl− than with gluconate, an impermeant anion. The rank order for anion stimulation of [35S]TBPS binding was Br−≧ SCN− > Cl−≧ NO−3 > I− > F− > gluconate. The EC50 value for Cl− enhancement of [35S]TBPS binding (160 mM) agreed well with the Km for Cl− influx via GABA‐gated channels (140 mM). These results indicate that TBPS acts as a GABA antagonist via direct blockade of neuronal Cl− channels. A minimum density of 6.5 × 104 chloride channels per neuron was obtained from TBPS binding at saturation.
Calcium‐Activated Proteolysis of Neurofilament Proteins in the Squid Giant Neuron
Authors: Gallant Paul E. , Pant Harish C. , Pruss Rebecca M. , Gainer Harold
Published: October 2006

Abstract: The phosphorylation and proteolysis of squid neurofilament proteins by endogenous kinase and calcium‐activated protease activities, respectively, were studied. When axoplasm was incubated in the presence of [γ‐32]ATP, most of the phosphate was incorporated into two neurofilament proteins: a 220‐kilodalton (NF‐220) and a high‐molecular‐weight (HMW) protein. When this phosphorylated axoplasm was subjected to endogenous calcium‐activated proteolysis, two significant phosphorylated fragments were generated, i.e., a soluble 110K fragment and a pelletable 100K fragment. Immunochemical and other analyses suggest that the pelletable 100K fragment contains the common helical neurofilament rod region and that the soluble 100K protein is the putative side arm of the NF‐220. In contrast, neither the HMW or the NF‐220 was detected in the region of the stellate ganglion which contains the cell bodies of the giant axon. However, this region did contain a number of proteins that were sensitive to calcium‐activated proteolysis and reacted with a monoclonal intermediate filament antibody. This intermediate filament antibody reacts with most of the axoplasmic proteins that copurify with neurofilaments, i.e., in the order of their intermediate filament antibody staining intensity, a 60K, 65K, 220K, and 74K protein. In the cell body preparation, the intermediate filament antibody labeled, in order of their staining intensity, a 65K, 60K, 74K, and 180K protein. In both the axoplasmic and cell body preparations, endogenous calcium‐activated proteolysis generated characteristic fragments that could be labeled with the anti‐intermediate filament antibody.