bluuebunny/crossref_metadata_2025 huggingface dataset:
Ethanol Does Not Modify Opiate‐Mediated Inhibition of Striatal Adenylate Cyclase
Authors: Hoffman Paula L. , Tabakoff Boris
Published: December 2006

Abstract: Ethanol increases the activity of “basal,” guanine nucleotide‐and dopamine‐stimulated adenylate cyclase in mouse striatum. In contrast, ethanol, in vitro, did not modify the inhibition of striatal adenylate cyclase activity by opiates (morphine or [D‐Ala2, D‐Leu5] enkephalin). Following chronic in vivo ethanol treatment of mice, there was also no change in the character of opiate inhibition of striatal adenylate cyclase activity. Since ethanol, in vitro, does decrease striatal opiate receptor binding, the results suggest that the changes in affinity detected by ligand binding studies are not relevant for receptor‐coupled adenylate cyclase activity, or that opiate receptor binding and opiate regulation of adenylate cyclase can be modulated independently. The selective effects of ethanol on systems that modulate adenylate cyclase activity may produce imbalances in neuronal function during in vivo ethanol exposure.
Changes of Uric Acid Level in Rat Brain After Focal Ischemia
Authors: Kanemitsu Hideaki , Tamura Akira , Sano Keiji , Iwamoto Takeo , Yoshiura Masahiko , Iriyama Keiji
Published: December 2006

Abstract: Changes of uric acid level in rat cerebral hemisphere after left middle cerebral artery (MCA) occlusion were studied by reversed‐phase HPLC with electrochemical detection. Uric acid level in the normal group was 2.98 nmol/g tissue. Uric acid concentration of the left hemisphere in the left MCA‐occluded group progressively increased after occlusion, and reached a maximum value of 67.26 nmol/g tissue 24 h after ischemia. Uric acid levels in the right hemisphere remained unchanged. Uric acid concentration of the left hemisphere in sham‐operated group was 9.29 nmol/g tissue 24 h after the operation.
Content of Quinolinic Acid and of Other Tryptophan Metabolites Increases in Brain Regions of Rats Used as Experimental Models of Hepatic Encephalopathy
Authors: Moroni F. , Lombardi G. , Carlà V. , Pellegrini D. , Carassale G. L. , Cortesini C.
Published: December 2006

Abstract: The content of the tryptophan metabolites quinolinic acid (QUIN), 5‐hydroxytryptamine (5‐HT), and 5‐hydroxyindoleacetic acid (5‐HIAA) was measured in various brain areas of rats bearing a portocaval anastomosis (PCA) for 4 weeks, using mass fragmentography or HPLC. In these animals, the content of the excitotoxic compound QUIN increased by 75% in the cortex and 125% in the cerebellum. The content of 5‐HT increased by 27% in the brainstem. No changes occurred in other brain areas. On the other hand, the content of 5‐HIAA increased by 66% in the cortex, 65% in the caudate, 64% in the hippocampus, 120% in the diencephalon, and 185% in the brainstem. Probenecid administration caused a larger increase of 5‐HIAA accumulation in various brain areas of PCA‐bearing rats than in those of sham‐operated controls. The cortical content of QUIN and 5‐HIAA increased after administration of ammonium acetate (7 mmol/kg), whereas an equimolar amount of sodium acetate was inactive. These results confirm that profound changes in the disposition of tryptophan occur in the brains of experimental animals used as models of hepatic encephalopathy. Furthermore, this study adds the excitotoxic compound QUIN to the list of molecules possibly involved in the pathogenesis of this brain disorder.
β‐Nerve Growth Factor Measurements in Mouse Serum
Authors: Lakshmanan J.
Published: December 2006

Abstract: Mouse serum β‐nerve growth factor (NGF) levels were measured using a newly developed competitive β‐NGF radioimmunoassay. The basal serum β‐NGF levels in male and female mice were consistently <2 ng/ ml when these animals were maintained in individual cages for at least 7 days before they were killed. However, in male mice, serum β‐NGF levels were significantly elevated when they were housed 5 per cage. The rise in serum β‐NGF levels, presumably mediated by intermale aggression, was confirmed by grouping previously isolated mice together in one cage for 20 min before they were killed. In all aggressive male mice, serum β‐NGF levels were elevated by two orders of magnitude. The β‐NGF radioimmunoassay values were also validated by a neurite outgrowth bioassay system using the serum of aggressive male mice. In summary, both measurement techniques confirm that mouse serum β‐NGF levels undergo marked changes depending on animal handling conditions.
Stimulation of the Serotonin Autoreceptor Prevents the Calcium‐Calmodulin‐Dependent Increase of Serotonin Biosynthesis in Rat Raphe Slices
Authors: Sawada Makoto , Nagatsu Toshiharu
Published: December 2006

Abstract: The role of the serotonin (5‐hydroxytryptamine) autoreceptor in the regulation of the activity of tryptophan hydroxylase was investigated in rat raphe slices. The activity of tryptophan hydroxylase was estimated by measuring the accumulation of 5‐hydroxytryptophan in the presence of inhibition of aromatic L‐amino acid decarboxylase using 3‐hydroxy‐4‐bromobenzyloxy‐amine by HPLC with fluorescence detection. Serotonin and its agonists N, N‐dimethyl‐5‐methoxytryptamine and 1‐(m‐chlorophenyl)‐piperazine reduced the formation of 5‐hydroxytryptophan to 50–60% at 10−5M. The effect of serotonin was reversed by 10−5M methiothepin. an antagonist of the serotonin autoreceptor. The calmodulin antagonists N‐(6‐aminohexyl)‐5‐chloro‐1 ‐naphthalenesul‐fonamide (W‐7) and N‐(6‐ammohexyl)‐1‐naphthalenesulfonamide (W‐5), dose‐dependently reduced the basal formation of 5‐hydroxytryptophan to 40–50% at 10−6 and 10−4M, respectively. W‐7 also reduced the activated formation by A‐23187 or dibutyryl cyclic AMP in a dose‐dependent manner. W‐7 had no effect on 5‐hydroxytryptophan formation reduced by serotonin at 10−5M. These results suggest that the role of the serotonin autoreceptor was related to the prevention of the calcium‐calmodulin‐dependent activation of tryptophan hydroxylase.
Correlation of Hexokinase Content and Basal Energy Metabolism in Discrete Regions of Rat Brain
Authors: Turek Thomas J. , Hawkins Richard A. , Wilson John E.
Published: December 2006

Abstract: Brain hexokinase (ATP:D‐hexose 6‐phospho‐transferase, EC 2.7.1.1) levels in seven regions of rat brain were estimated by photometric measurement of im‐munofluorescence in cryostat‐cut sections. When compared with basal rates of glucose metabolism in these regions, estimated by the 6‐[14C]glucose method, a significant correlation was observed. Thus, hexokinase content reflects metabolic energy demands.
Dopaminergic Inhibition of Prolactin Release and Calcium Influx Induced by Neurotensin in Anterior Pituitary Is Independent of Cyclic AMP System
Authors: Memo M. , Castelletti L. , Missale C. , Valerio A. , Carruba M. , Spano P. F.
Published: December 2006

Abstract: The present study demonstrates that 3,4‐dihydroxyphenylethylamine (DA, dopamine) prevents neurotensin (NT) stimulation of both prolactin (PRL) release and calcium influx by interacting with specific receptors that are functionally linked to calcium channels. As shown by the studies with dispersed cells from rat anterior pituitary, the pharmacology of the control of PRL release and calcium influx, both induced by NT, was found to be typical of a DAergic process. This was demonstrated (1) by the order of potency of agonists in inhibiting PRL release and calcium influx (DA > epinephrine > norepinephrine isoproterenol); (2) by the high affinity of antagonists such as haloperidol and fluphenazine for this process; and (3) by the high degree of stereoselectivity of sulphide. Specific D2 receptor agonists, such as bromocriptine and lisuride, and the specific D2 receptor antagonist (‐)‐sulpiride were found to be highly potent on the DA receptors negatively coupled with calcium channels and PRL release. DA was found to lack the capacity to change the influx of calcium induced by either the sodium channel activator veratridine or high extracellular potassium levels, thus indicating a specific action of this amine on calcium channels sensitive to NT. In a range of concentrations that are effective in inhibiting either the calcium influx or the PRL release, both induced by NT, DA did not alter the cyclic AMP generating system. DA (from 1.0 nM to 50 nM) did not affect adenylate cyclase activity in rat pituitary gland homogenates and did not modify intracellular cyclic AMP levels in pituitary cells. The putative correlation between DA suppressing activity of the NT effects and the adenylate cyclase system was verified in pertussis toxin‐treated pituitary cells. At the time that DA receptors negatively coupled to inhibition of adenylate cyclase were not operative, DA was still fully effective in preventing NT stimulation of PRL release. The lack of effect of low concentrations of DA on both basal adenylate cyclase activity and cyclic AMP content in pituitary cells, together with their capacity to inhibit the NT effects in pertussis toxin‐treated cells, suggest that DA may act independently of cyclic AMP in the control of PRL secretion. These observations lead to the hypothesis of the existence of two inhibitory mechanisms for DA in mammotrophs.
A Ganglioside Species (GD1α) Migrates at a Slow Rate and CMP‐Sialic Acid Severalfold Faster in Xenopus Sciatic Nerve: Fluorographic Demonstration
Authors: Igarashi Michihiro , Komiya Yoshiaki , Kurokawa Masanori
Published: December 2006

Abstract: The ninth dorsal root ganglion of adult Xenopus laevis was labeled with N‐acetyl‐D‐[6‐3H]mannosamine, and intraaxonal migration of gangliosides was examined by analysis of the chloroform/methanol extract of each of 5‐mm consecutive nerve segments by TLC coupled with fluorography. A unique disialoganglioside (GD1α), which amounted to up to 83% of the total ganglioside in this nerve, migrated at 1–2 mm/day at 15°C. This contrasts with the rapid transport of other ganglioside species previously reported in the optic systems of goldfish, rabbits, chickens, and rats. Fluorographic analysis also revealed a trichloro‐acetic acid‐soluble substance migrating at a velocity of 8 mm/day at 15°C. The substance was considered to be CMP‐sialic acid on the basis of observations that it comigrates with authentic CMP‐N‐acetylneuraminic acid in TLC developed with two different solvent systems, it is very labile to weak acid but resistant to neuraminidase from Vibriocholerae, it is converted to N‐acetylmannosamine when treated first with weak acid and subsequently with N‐acetyl‐neuraminic acid aldolase, and it has a β‐sialosyl group in its structure. Because CMP‐sialic acid is believed to be the sole sialosyl donor in the cells, its migration in axons toward terminals, together with the previous demonstration of sialyl‐transferase activity in the synaptosomal plasma membrane, strongly supports the possibility that sialosylation of gangliosides and probably of other sialoglycoproteins is not confined to the Golgi apparatus, but can also occur after the compounds are committed to axonal transport.
A Nerve Growth Factor‐Sensitive S6 Kinase in Cell‐Free Extracts from PC 12 Cells
Authors: Matsuda Yuzuru , Nakanishi Nobuo , Dickens Geneva , Guroff Gordon
Published: December 2006

Abstract: Soluble extracts from nerve growth factor (NGF)‐stimulated PC 12 cells prepared by alkaline lysis show a two‐to 10‐fold greater ability to phosphorylate the 40S ribosomal protein S6 than do extracts from control cells. The alkaline lysis method yields a preparation of much higher specific activity than does sonication. Half‐maximal incorporation of 32P from [32P]ATP into S6 occurred after 4–7 min of NGF treatment. The partially purified NGF‐sensitive S6 kinase has a molecular weight of 45,000. It is not inhibited by NaCl, chlorpromazine, or the specific inhibitor of cyclic AMP (cAMP)‐dependent protein kinase, nor is it activated by addition of diolein plus phosphatidylserine. Trypsin treatment of either crude extracts or partially purified S6 kinase from control or NGF‐treated cells was without effect. These data suggest that the S6 kinase stimulated by NGF is neither cAMP‐dependent protein kinase or protein kinase C nor the result of tryptic activation of an inactive proenzyme. Treatment of intact cells with dibutyryl cAMP or 5′‐N‐ethylcarboxamideadenosine also increases the subsequent cell‐free phosphorylation of S6. This observation suggests that cAMP‐dependent protein kinase may be involved in the phosphorylation of S6 kinase.
High‐Affinity Receptor Sites and Rapid Proteolytic Inactivation of Neurotensin in Primary Cultured Neurons
Authors: Checler Frédéric , Mazella Jean , Kitabgi Patrick , Vincent Jean‐Pierre
Published: December 2006

Abstract: The present article describes the interaction of neurotensin with specific receptors in pure primary cultured neurons and the mechanisms by which this peptide is inactivated by these cells. Neurotensin binding sites are not detectable in nondifferentiated neurons and appear during maturation. The binding at 37°C of [monoiodo‐Tyr3 neurotensin to monolayers of neurons 96 h after plating is saturable and characterized by a dissociation constant of 300 pM and a maximal binding capacity of 178 fmol/mg of protein. The binding parameters as well as the specificity of these receptors toward neurotensin analogues reveal close similarities between the binding sites present in primary cultured neurons and those described in other membrane preprations or cells. Neurotensin is rapidly degraded by primary cultured neurons. The sites of primary inactivating cleavages are the Pro7‐Arg8, Arg8‐Arg9, and Pro10‐Tyr11 bonds. Proline endopeptidase is totally responsible for the cleavage at the Pro7‐Arg8 bond and contributes to the hydrolysis mainly at the Pro10‐Tyr11 site. However, the latter breakdown is also generated by a neurotensin‐degrading neutral metallopeptidase. The cleavage at the Arg8‐Arg9 bond is due to a peptidase that can be specifically inhibited by N‐[1 (R, S)‐carboxy‐2‐phenylethyl]‐alanyl‐alanyl‐phenylalanyl‐p‐aminobenzoate. The secondary processing occurring on neurotensin degradation products are: (1) a bestatin‐sensitive aminopeptidasic conversion of neurotensin, 11‐13 to free Tyr11, and (2) a rapid cleavage of neurotensin8‐13 by proline endopeptidase. A model for the inactivation of neurotensin in primary cultured neurons is proposed and compared to that previously described for purified rat brain synaptic membranes.
Stimulatory Effects of Growth Hormone and Thyroxine on the Concentration of Gangliosides in the Snell Dwarf Cerebrum
Authors: Noguchi Tetsuya , Sugisaki Tetsuro
Published: December 2006

Abstract: The concentration of gangliosides in the Snell dwarf mouse cerebrum was monitored from postnatal day 5 to day 40. In the dwarf cerebrum, the concentration of total gangliosides increased up to postnatal day 20 and then stopped, whereas in the control cerebrum, it continued to increase up to postnatal day 40. At postnatal day 40, the ganglioside level in the dwarf cerebrum was 70% of that in the control cerebrum. Among the ganglioside species, the concentrations of GM4, GM2, GM1, GD1a, GD3, GD1b, GT1b, and GQ1b were significantly lower in the dwarf cerebrum than in the controls at postnatal day 40. The reduced concentrations of ganglioside species GM2, GD1a, GD3, GD1b, and GQ1b, were completely restored by administration of bovine growth hormone (GH) during the first 20 days of postnatal life. The reduced concentration of the GM1 and GM4 species were most efficiently restored by administration of bovine GH plus thyroxine (T4) during the second 20 days of postnatal life. These results indicate that the lower ganglioside concentrations in the dwarf cerebrum can be elevated by hormone therapy and that there exist distinct GH and T4 actions on the enzymes participating in ganglioside metabolism.
Superoxide Radical‐Mediated Alteration of Synaptosome Membrane Structure and High‐Affinity γ‐[14C]Aminobutyric Acid Uptake
Authors: Debler Edmund A. , Sershen Henry , Lajtha Abel , Gennaro Joseph F.
Published: December 2006

Abstract: Mouse cortical synaptosomal structure and function are altered when exposed to hypoxanthine/xanthine oxidase (HPX/XOD)‐generated active oxygen/free radical species. The structure of both the synaptic vesicle and plasma membrane systems are altered by HPX/XOD treatment. The alteration of synaptic vesicle structure is exhibited by a significant increase in the cumulative length of nonsynaptic vesicle membrane per nerve terminal. With respect to the nerve terminal plasma membrane, the length of the perimeter of the synaptosome is increased as the membrane pulls away from portions of the terminal in blebs. The functional lesion generated by HPX/XOD treatment results in a reduction in selective high‐affinity γ‐[14C]aminobutyric acid (GABA) uptake. Kinetic analysis of the reduction in high‐affinity uptake reveals that the V.max is significantly altered whereas the Km is not. Preincubation with specific active oxygen/free radical scavengers indicates that the superoxide radical is directly involved. This radical, most probably in the protonated perhydroxyl form, initiates lipid peroxidative damage of the synaptosomal membrane systems. Low‐affinity [14C]GABA transport is unaltered by the HPX/XOD treatment. The apparent ineffectiveness of free radical exposure on low‐affinity [14C]GABA transport coupled with its effectiveness in reducing high‐affinity transport supports the idea that two separate and different amino acid uptake systems exist in CNS tissue, with the high‐affinity being more sensitive (lipid‐dependent) and/or more energy‐dependent (Na+, K+‐ATPase) than the low‐affinity system.
Convulsive Activity of α‐Guanidinoglutaric Acid and the Possible Involvement of 5‐Hydroxytryptamine in the α‐Guanidinoglutaric Acid‐Induced Seizure Mechanism
Authors: Shiraga Hiroshi , Hiramatsu Midori , Mori Akitane
Published: December 2006

Abstract: α‐Guanidinoglutaric acid (α‐GGA) was first found in cobalt‐induced epileptogenic focus tissue in the cerebral cortex of cats. We examined the effect of α‐GGA on the electroencephalogram and on the brain 5‐hydroxytryptamine (5‐HT) level after intraventricular administration into rats. Sporadic low‐voltage spikes appeared 4 min after the administration of α‐GGA. Spikes increased in voltage 6 min after the administration. Multiple spikes appeared 10 min after the administration, and they reached maximal frequency 30 min after the administration. The epileptic discharges disappeared 100 min after the administration. The 5‐HT level increased in the right and left cortices 3 min after the administration. The 5‐HT level decreased in the mid‐brain 5 min after the administration and subsequently in all regions of the brain 10 min after the administration. No change in the 5‐HT level was found 30 min and 100 min after the administration. These results show that α‐GGA induces epileptic seizures in rats after intraventricular administration. The results also suggest that α‐GGA‐induced seizures are associated with abnormal serotonergic function and that they are initiated by a decrease in the 5‐HT level.
Biochemical Correlates of Myelination in Brain and Spinal Cord of Mice Heterozygous for the Jimpy Gene
Authors: Benjamins Joyce A. , Studzinski Diane M. , Skoff Robert P.
Published: December 2006

Abstract: Brain and spinal cord of female mice heterozygous for the jimpy gene were analyzed during development for activity of ceramide galactosyl transferase (CGT) and for levels of myelin basic protein (MBP). CGT activity was low at 13–14 days in brains of heterozygous jimpy females but showed normal levels by 31 –36 days, in agreement with our earlier study of this enzyme. In cord, CGT activity was normal or slightly above normal at all ages studied, from 13–14 days into adulthood. In both brain and cord, decreased levels of MBP were observed at 13 days; by 100 days, amounts of MBP approached normal levels. Proven female carriers of the jimpy gene also showed normal levels of CGT activity, MBP, and isolated myelin at 200–250 days of age in both brain and cord. These biochemical findings agree with previous morphologic measurements in cord demonstrating deficits in myelin at early ages but compensation by 100 days. Our results show that compensation occurs earlier in cord than in brain and that levels of MBP show a closer correlation than CGT activity with amounts of myelin, as measured by either morphometric analysis or direct isolation.
5,5‐Diphenylhydantoin Antagonizes Neurochemical and Behavioral Effects of p, p'‐DDT but Not of Chlordecone
Authors: Tilson Hugh A. , Hudson P. M. , Hong J. S.
Published: December 2006

Abstract: Rats were given 75 mg/kg of 5,5‐diphenylhydantoin (phenytoin) or vehicle 30 min prior to 75 mg/kg of 1, 1, 1‐trichloro‐bis(p‐chlorophenyl)ethane (p, p'‐DDT) (p.o.) or chlordecone (i.p.) and tremor was measured 12 h later. Rats were then killed, and regional brain levels of biogenie amines and their acid metabolites and amino acids were determined. Pretreatment with phenytoin significantly attenuated the tremor produced by p, p'‐DDT but enhanced that produced by chlordecone. p, p'‐DDT had significant effects on the levels of asparate, glutamate, 5‐hydroxyindoleacetic acid (5‐HIAA), and 3‐methoxy‐4‐hydroxyphenylglycol (MHPG), whereas chlordecone increased glycine, 5‐HIAA, and MHPG levels. Pretreatment with phenytoin blocked p.p'‐DDT‐induced increases of aspartate in the brainstem and spinal cord, 5‐HIAA in the hippocampus, and MHPG in the brainstem and hypothalamus. Phenytoin significantly enhanced chlordecone‐induced increases of MHPG in the brainstem. These data indicate that organo‐chlorine‐induced increases in noradrenergic activity in the brainstem and spinal cord may be directly related to the tremorigenic effects of these chemicals.
Effects of GABAergic Drugs In Vivo on High‐Affinity Choline Uptake In Vitro in Mouse Hippocampal Synaptosomes
Authors: Miller J. A. , Richter J. A.
Published: December 2006

Abstract: The effects of several γ‐aminobutyric acid (GABA)‐ergic drugs on sodium‐dependent high‐affinity choline uptake (HACU) were investigated in the hippocampus. HACU was measured in vitro after in vivo administration of the drug to mice. HACU was inhibited by those drugs that enhance GABA transmission. The convulsant 3‐mercaptopropionic acid, which decreases GABA levels, stimulated HACU. From these results and previous find ings, it appears that GABA mediates a tonic inhibitory effect on the septal‐hippocampal cholinergic system.
Localization of the Phosphorylation Sites for Different Kinases in the Microtubule‐Associated Protein MAP2
Authors: Hernández Maria Angeles , Wandosell Francisco , Avila Jesús
Published: December 2006

Abstract: The phosphorylation of microtubule‐associated protein 2 (MAP2) by four different kinases was studied in vitro to determine whether MAP2 is phosphorylated in its tubulin binding region or in the microtubule projection portion. Fragments corresponding to both regions of MAP2 were produced not only by chymotrypsin or trypsin digestion, but also using pepsin, a broad chain‐specificity protease, a result supporting previous notions of the two‐domain structure of MAP2. The position of these two functional domains was determined with respect to the carboxy terminal of the molecule, by labeling MAP2 exclusively at the carboxy terminal and subjecting it to pepsin digestion. The results suggested that the projection region of MAP2 contained the carboxy terminal of the protein. A phosphorylation map was constructed by subjecting phosphorylated MAP2 to enzymatic digestion using Staphylococcus aureus V8 protease or to chemical cleavage using N‐chlorosuccinimide. The results indicated that all four kinases phosphorylated MAP2 in a 42‐kilodalton peptide that contained the tubulin binding region but differed in the level and localization of the sites at which they phosphorylated the projection of MAP2.
[3H]Neurokinin B and 125I‐Bolton Hunter Eledoisin Label Identical Tachykinin Binding Sites in the Rat Brain
Authors: Bergstrom L. , Torrens Y. , Saffroy M. , Beaujouan J. C. , Lavielle S. , Chassaing G. , Morgat J. L. , Glowinski J. , Marquet A.
Published: December 2006

Abstract: [3H]Neurokinin B ([3H]NKB) of high specific activity (75 Ci/mmol) was synthesized for study of its binding to crude synaptosomes from the rat cerebral cortex. The specific binding of [3H]NKB (75% of total binding) was temperature dependent, saturable, and reversible. Scatchard analyses and Hill plots showed the existence of a single population of noninteracting binding sites (KD= 4.3 nM; Bmax= 123 fmol/mg of protein). Competition studies indicated the following rank order of potencies among tachykinins: NKB > eledoisin (E) > kassinin > physalaemin > neurokinin A (NKA) > substance P (SP), a result suggesting that NKB might be the endogenous ligand for [3H]NKB binding sites. It is of interest that 127I‐Bolton Hunter (BH) NKA (127I‐BHNKA) was much more potent than NKA in inhibiting the specific binding of [3H]NKB, which raises certain questions concerning the use of 125I‐BHNKA as a Iigand for NKA binding sites in the brain. These results, as well as those obtained with different SP analogues, show a close similarity to those obtained previously with 125I‐BHE binding to cortical synaptosomes. This suggested that the two ligands labeled identical binding sites. In addition, using either [3H]NKB or 125I‐BHE as ligands, similar displacement curves were obtained with increasing concentrations of NKB and 127I‐BHE. The similarity of the [3H]NKB and 125I‐BHE binding sites was further confirmed by comparison of their localization on rat brain sections by autoradiography. The distribution of binding sites for [3H]NKB and 125I‐BHE was identical throughout the brain, and the highest density of binding sites for the two ligands was found in layers IV and V of the cerebral cortex, the paraventricular nucleus of the hypothalamus (magnocellular part), and the ventral tegmental area.
Destinations of Some Fast‐Transported Proteins in Sensory Neurons of Bullfrog Sciatic Nerve
Authors: Rulli R. Dino , Wilson David L.
Published: December 2006

Abstract: Many characteristics of proteins that are fast axonally transported have been described, but the destinations of most within the neuron remain unknown. We have studied the destinations of some fast‐transported proteins in sensory neurons of the bullfrog sciatic nerve, specifically to determine which may be deposited in axons and which may be destined for more distal, possibly terminal, areas. Dorsal root ganglia were pulse‐labeled with [35S]methionine in vitro, following which they were separated from the sciatic nerve. After additional periods of transport, radioactive proteins from two areas of the nerve were separated by two‐dimensional polyacrylamide gel electrophoresis and used to develop x‐ray film. The first area contained the wavefront of transported radioactivity (wavefront region), whereas the second area was taken from nerve through which the wavefront had already passed (plateau region). The amount of radioactivity in certain fast‐transported protein species from each area was determined by computer analysis of digitized video images of fluorographs. Certain proteins were preferentially left behind the wavefront and, therefore, may supply axon and possibly other nerve components, whereas other proteins were found almost exclusively in the wavefront and, hence, may supply more distal, possibly terminal, areas.
Increased Diacylglycerol Levels Inhibit [20‐3H]Phorbol 12, 13‐Dibutyrate Binding and the Glucocorticoid‐Mediated Increase in Glycerol Phosphate Dehydrogenase Levels in C6 Rat Glioma Cells
Authors: Bressler Joseph
Published: December 2006

Abstract: I examined whether the phorbol ester‐mediated inhibition of glycerol 3‐phosphate dehydrogenase (GPDH) induction could be mimicked by raising the cellular diacylglycerol levels. Phorbol ester tumor promoters and diacylglycerols activate protein kinase C. An increase in radiolabeled diacylglycerol levels in C6 rat glioma cells was observed when cells were prelabeled overnight with [3H]arachidonic acid and treated with either phospholipase C (Clostridium perfringens) or 2‐bromooctanoate. The increase was dose dependent. The diacylglycerols competed with [20‐3H]phorbol 12, 13‐dibutyrate in binding to the phorbol ester receptor. A Scatchard analysis of the binding of cells treated with 0.1 unit/ml of phospholipase C demonstrated that the inhibition was mainly due to a decrease in binding affinity and not in the total number of binding sites. 2‐Bromooctanoate and phospholipase C, but not the synthetic diacylglycerol l‐oleoyl 2‐acetyl glycerol, inhibited the glucocorticoid induction of GPDH levels. Boiled phospholipase C, phospholipase A2, or phospholipase D was ineffective in inhibiting induction, a result suggesting that the inhibition was not due to nonspecific membrane perturbation. Thus, inhibition of the glucocorticoid‐mediated increase in GPDH induction is most likely mediated by protein kinase C, and not by an alternate phorbol ester receptor.
Activation of Tyrosine Hydroxylase in PC12 Cells by the Cyclic GMP and Cyclic AMP Second Messenger Systems
Authors: Roskoski Robert , Roskoski Laura M.
Published: December 2006

Abstract: Tyrosine hydroxylase, the rate‐limiting enzyme in catecholamine biosynthesis, is subject to regulation by a variety of agents. Previous workers have found that cyclic AMP‐dependent protein kinase and calcium‐stimulated protein kinases activate tyrosine hydroxylase. We wanted to determine whether cyclic GMP might also be involved in the regulation of tyrosine hydroxylase activity. We found that treatment of rat PC12 cells with sodium nitroprusside (an activator of guanylate cyclase), 8‐bromocyclic GMP, forskolin (an activator of adenylate cyclase), and 8‐bromocyclic AMP all produced an increase in tyrosine hydroxylase activity measured in vitro or an increased conversion of [14C]tyrosine to labeled catecholamine in situ. Sodium nitroprusside also increased the relative synthesis of cyclic GMP in these cells. In the presence of MgATP, both cyclic GMP and cyclic AMP increased tyrosine hydroxylase activity in PC12 cell extracts. The heat‐stable cyclic AMP‐dependent protein kinase inhibitor failed to attenuate the activation produced in the presence of cyclic GMP. It eliminated the activation produced in the presence of cyclic AMP. Sodium nitroprusside also increased tyrosine hydroxylase activity in vitro in rat corpus striatal synaptosomes and bovine adrenal chromaffin cells. In all cases, the cyclic AMP‐dependent activation of tyrosine hydroxylase was greater than that of the cyclic GMP‐dependent second messenger system. These results indicate that both cyclic GMP and cyclic AMP and their cognate protein kinases activate tyrosine hydroxylase activity in PC12 cells.
Stimulation‐Evoked Ca2+ Fluxes in Cultured Bovine Adrenal Chromaffin Cells Are Enhanced by Forskolin
Authors: Morita Katsuya , Dohi Toshihiro , Kitayama Shigeo , Kyama Yutaka , Tsujimoto Akira
Published: December 2006

Abstract: Forskolin, 1 μM, increased acetylcholine (ACh)‐stimulated 45Ca uptake by chromaffin cells. The stimulatory effects of forskolin decreased with increasing concentration of ACh. The attenuation of the effect of forskolin on 45Ca uptake as a function of ACh concentration correlated well with changes in the forskolin effect on ACh‐evoked cate‐cholamine (CA) release. Forskolin increased excess KCl‐and veratrine‐evoked CA release and 45Ca uptake. Forskolin by itself stimulated 45Ca efflux and enhanced ACh‐, excess KCl‐, and veratrine‐stimulated 45Ca efflux. High doses of forskolin inhibited both ACh‐evoked 45Ca uptake and CA release. The inhibitory action of forskolin was specific to receptor‐mediated response because excess KCl‐and vera trine‐stimulated 45Ca uptake and CA release were not inhibited. Forskolin, 0.3–30 μM, dose‐dependently increased caffeine‐stimulated CA release and 45Ca efflux in the absence of Ca2+ in the medium, and the effects were mimicked by dibutyryl cyclic AMP. These results suggest that cyclic AMP increases stimulation‐induced CA release by enhancing calcium uptake across the plasma membrane and/or altering calcium flux in an intracellular calcium store.
Synaptosomal Membrane‐Bound Form of Endopeptidase‐24.15 Generates Leu‐Enkephalin from Dynorphin 1‐8, α‐ and β‐Neoendorphin, and Met‐Enkephalin from Met‐Enkephalin‐Arg6‐Gly7‐Leu
Authors: Acker Gwendolyn R. , Molineaux Christopher , Orlowski Marian
Published: December 2006

Abstract: Brain contains a membrane‐bound form of endopeptidase‐24. 15, a metalloendopeptidase predominantly associated with the soluble protein fraction of brain homogenates. Subcellular fractionation of the enzyme in rat brain showed that 20–25% of the total activity is associated with membrane fractions including synaptosomes. Solubilization of the enzyme from synaptosomal membranes required the use of detergents or treatment with trypsin. The specific activity of the enzyme in synaptosomal membranes measured with tertiary‐butoxycarbonyl‐Phe‐Ala‐Ala‐Phe‐p‐aminobenzoate as substrate was higher than that of endo‐peptidase‐24.11 (“enkephalinase”), a membrane‐bound zinc‐metalloendopeptidase believed to function in brain neuropeptide metabolism. Purified synaptosomal membranes converted efficiently dynorphini1‐8, α‐and β‐neoen‐dorphin into leucine enkephalin and methionine‐enkepha‐Iin‐Arg6‐Gly7‐Leu8 into methionine enkephalin in the presence of captopril, bestatin, and N‐[l‐ (R, S)‐carboxy‐2‐phenylethyl]‐Phe‐p‐aminobenzoate, inhibitors of angiotensin converting enzyme (EC 3.4.15.1), aminopeptidase (EC 3.4.11.2), and membrane‐bound metalloendopeptidase (EC 3.4.24.11), respectively. The conversion of enkephalin‐containing peptides into enkephalins was virtually completely inhibited by N‐[l‐ (R, S)‐carboxy‐2‐phenylethyl]‐Ala‐Ala‐Phe‐p‐aminobenzoate, a specific active‐site‐directed inhibitor of endopeptidase‐24.15, indicating that this enzyme was responsible for the observed interconversions. The data indicate that synaptosomal membranes contain enzymes that can potentially generate and degrade both leucine‐and methionine‐enkephalin.
Stimulation of Melatonin Synthesis in Ovine Pineals In Vitro
Authors: Morgan P. J. , Williams L. M. , Lawson W. , Riddoch G.
Published: December 2006

Abstract: Static and superfused pineal slices (750 μm) have been used to study the control of melatonin synthesis by ovine pineals. Static incubates show a time‐dependent accumulation of melatonin in the medium; this is significantly increased by stimulation with norepinephrine (NE) (10−5M), reaching 300% above control levels after 4 h. Perifused pineal slices show a rapid rise in melatonin release within 12–18 min in response to NE stimulation. This reaches a 3.5–4.5‐fold increase in melatonin released within 30 min. Withdrawal of NE is associated with a rapid return to prestimulated levels within 12–18 min. These time‐course characteristics compare favorably to those changes seen in vivo. The formation of [14C]melatonin from [14C]‐tryptophan shows a linear increase with time. In the presence of NE (10−5M), the rate of synthesis is increased, albeit after an initial time lag of at least 30 min. The latter may reflect an N‐acetyltransferase‐independent mechanism of synthesis and release. In static incubations, propranolol (10−5M) inhibited NE‐induced melatonin production by about 60%. but prazosin (10−5M) had no effect. As dibutyryl cyclic AMP (10−3M) stimulated melatonin production, it is concluded that β‐receptors are of primary importance to the control of melatonin production, as in the rat. The role of α1‐receptors is less clear, but the stimulatory action of phorbol 12‐myristate 13‐acetate on melatonin release implicates a receptor linked to phosphatidylinositol turnover.
Antioxidant Action and Photosensitizing Effects of Three Different Chlorpromazines
Authors: Bindoli Alberto , Rigobello Maria Pia , Favel Anne , Galzigna Lauro
Published: December 2006

Abstract: Chlorpromazine inhibits by about 60% the lipid peroxidation stimulated by Fe2+/ascorbate in liposomes and the lipid peroxidation stimulated by cumene hydro‐peroxide in microsomes. Under the same conditions, two new synthetic derivatives of chlorpromazine, i.e., a N‐benzoyloxymethylchlorpromazine and a N‐pivaloyloxymethylchlorpromazine, induce no more than a 20% inhibition. On the other hand, when the different chlorpromazines are entrapped in liposomes and subsequently irradiated with near‐UV light, they act as photosensitizing agents giving rise to lipid peroxidation. The latter is quite extensive in the presence of chlorpromazine or N‐pivaloyloxymethylchlorpromazine, whereas it is drastically lower in the presence of N‐benzoyloxymethylchlorpromazine. The N‐benzoyloxymethylchlorpromazine molecule, despite its low photodyn‐amic effect, retains its neuroleptic properties. The possible mechanisms of the antioxidant and prooxidant actions of these compounds are discussed.
Presence of Base‐Exchange Activity in Rat Brain Nerve Endings: Dependence on Soluble Substrate Concentrations and Effect of Cations
Authors: Holbrook P. G. , Wurtman R. J.
Published: December 2006

Abstract: The calcium‐dependent, energy‐independent incorporations of 14C‐labeled bases, choline, ethanolamine, and serine, into their corresponding membrane phospholipids, phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine, were compared in microsomes and in subcellular fractions prepared from a lysed crude mitochondrial (P2) pellet of whole rat brain. When activities were measured in the presence of an extracellular (1.25 mM) concentration of Ca2+, recovered activities were highest in the microsomal fraction, although substantial activity remained associated with the P2 homogenate even after repeated washing of the pellet. When this washed P2 homogenate was subfractionated, enrichment of all three exchange activities was obtained only in a fraction that was fivefold enriched over the homogenate and sevenfold enriched over the microsomal fraction in Na+, K+‐ATPase, a plasma membrane marker. This strongly suggests that the base‐exchange enzymes are normal constituents of synaptosomal plasma membranes. The three exchange activities were measured in synaptosomes prepared from whole rat brain in the presence of various substrate (base) concentrations, and kinetic constants were calculated. The Vmax values for choline, ethanolamine, and serine exchange were, respectively, 1.27 ± 0.09, 1.60 ± 0.17, and 0.56 ± 0.06 nmol/mg of protein/h; the respective Km(apparent) values were 241 ± 29, 65 ± 18, and 77 ± 22 μM. Endogenous levels of the three bases, choline, ethanolamine, and serine, in whole (microwaved) rat brains were 20 ± 8, 78 ± 28, and 639 ± 106 nmol/g, respectively. That ethanolamine and serine incorporations had lower Km values than choline incorporation suggests that these bases are preferentially incorporated into their respective phospholipids. Moreover, that endogenous choline levels are well below those needed for enzyme saturation suggests that the incorooration of choline into phosphatidylcholine via this pathway may be sensitive to transitory increases in free choline concentration (such as occur intrasynaptically after acetyl‐choline is released and hydrolyzed). Various polyvalent cations (Mg2+, Ba2+, Ni2+, Co2+, and La3+) inhibited synaptosomal base‐exchange activity with a rank order of potency similar to their ability to block calcium channels.
Purification and Characterization of Two Soluble C1−‐Activated Arginyl Aminopeptidases from Human Brain and Their Endopeptidase Action on Neuropeptides
Authors: McDermott J. R. , Mantle D. , Lauffart B. , Gibson A. M. , Biggins J. A.
Published: December 2006

Abstract: Two closely related Cl− ‐activated arginyl aminopeptidases (I and II) were purified from a soluble extract of postmortem human cerebral cortex by anion‐exchange chromatography and preparative gel electrophoresis. The electrophoretic mobility of II was approximately 80% that of I; the molecular mass of both enzymes was approximately 70 kilodaltons (kDa) (gel filtration). The aminopeptidase action of 1 and II on aminoacyl‐7‐amido‐4‐methyl‐coumarin (AMC) substrates was restricted to the Arg and Lys derivatives. Both enzymes had significant endopeptidase activity, hydrolysing several biologically active peg tides including neurotensin, bradykinin, angiotensin‐I, substance P, luliberin, and somatostatin at internal bonds. Other peptides [Leu‐enkephalin, proctolin, thyroliberin, adrenocorticotropin18–39 (ACTH18–39), ACTH11–24, and dynorphin (1–13)] were not appreciably hydrolysed. The amino‐ and endopeptidase activities had pH optima at 6.5 and 7, respectively, and were both inhibited by metal ion chelators and sulphydryl group blocking agents. The ami‐nopeptidase activity was stimulated 20‐fold by Cl− ions, whereas the endopeptidase activity was unaffected by the latter. Km values for neurotensin degradation were 20 μM (I) and 37 μM (II) and for Arg‐AMC hydrolysis they were 167 μM (I) and 125 μM (II). The endopeptidase activity was not inhibited by the aminopeptidase inhibitors arphame‐nine or bestatin (IC50= 9 nM and 0.1 μM, respectively, with Arg‐AMC substrate).
Levels of Catechols in Epileptogenic and Nonepileptogenic Regions of the Human Brain
Authors: Goldstein David S. , Nadi N. Suzan , Stull Robin , Wyler Allen R. , Porter Roger J.
Published: December 2006

Abstract: Recent reports about tyrosine hydroxylase and α1‐adrenoceptors in epileptic foci have suggested increased regional catecholaminergic activity, which may serve a compensatory, inhibitory role. We measured levels of catechols, including the precursor 3,4‐dihydroxyphenylalanine (Dopa) and the catecholamines dopamine (DA) and nor‐epinephrine (NE), in surgically removed foci identified by electrocorticography and in nonepileptogenic sites from 23 patients with intractable temporal lobe epilepsy. The following values (mean ± 1 SD) were obtained: DOPA = 142 ± 60 ng/g of protein in the focus vs. 115 ± 39 ng/g in the nonfocus (p < 0.01); DA = 168 ± 85 vs. 106 ± 54 ng/g (p < 0.001); and NE = 267 ± 117 vs. 181 ± 80 ng/g (p < 0.001). The results are consistent with increased catecholaminergic activity in epileptic foci.
Nerve Growth Factor Induces Na+, K+‐ATPase in a Nerve Cell Line
Authors: Inoue Nobuo , Matsui Hideo , Hatanaka Hiroshi
Published: December 2006

Abstract: The effects of nerve growth factor (NGF) on induction of Na+, K+‐ATPase were examined in a rat pheochromocytoma cell line, PC12h. Na+, K+‐ATPase activity in a crude particulate fraction from the cells increased from 0.37 ± 0.02(n = 19) to 0.55 ± 0.02 (n = 20) (means ± SEM, μmol Pi/min/mg of protein) when cultured with NGF for 5–11 days. The increase caused by NGF was prevented by addition of specific anti‐NGF antibodies. Epidermal growth factor and insulin had only a small effect on induction of Na+, K+‐ATPase. A concentration of basic fibroblast growth factor three times higher than that of NGF showed a similar potency to NGF. The molecular form of the enzyme was judged as only the α form in both the untreated and the NGF‐treated cells by a simple pattern of low‐affinity interaction with cardiotonic steroids: inhibition of enzyme activity by strophanthidin (Ki∼ 1 mM) and inhibition of Rb+ uptake by ouabain (Ki∼ 100μM). As a consequence, during differentiation of PC12h cells to neuron‐like cells, NGF increases the α form of Na+, K+‐ATPase, but does not induce the α(+) form of the enzyme, which has a high sensitivity for cardiotonic steroid and is a characteristic form in neurons.
4‐(O‐Benzylphenoxy)‐N‐Methylbutylamine (Bifemelane) and Other 4‐(O‐Benzylphenoxy)‐N‐Methylalkylamines as New Inhibitors of Type A and B Monoamine Oxidase
Authors: Naoi Makoto , Nomura Yoshio , Ishiki Ryoji , Suzuki Hiroko , Nagatsu Toshiharu
Published: December 2006

Abstract: 4‐(O‐Benzylphenoxy)‐N‐methylbutylamine (Bifemelane, BP‐N‐methylbutylamine), a new psychotropic drug, was found to inhibit monoamine oxidase (MAO) in human brain synaptosomes. It inhibited type A MAO (MAO‐A) competitively and type B (MAO‐B) noncompetitively. BP‐N‐methylbutylamine had a much higher affinity to MAO‐A than an amine substrate, kynuramine, and it was a more potent inhibitor of MAO‐A than of MAO‐B. The Ki values of MAO‐A and ‐B were determined to be 4.20 and 46.0 μM, respectively, while the Km values of MAO‐A and ‐B with kynuramine were 44.1 and 90.0 μM, respectively. The inhibition of MAO‐A and ‐B by BP‐N‐methylbutylamine was found to be reversible by dialysis of the incubation mixture. MAO‐A in human placental and liver mitochondria and in a rat clonal pheochromocytoma cell line, PC12h, was inhibited competitively by BP‐N‐methylbutylamine, while MAO‐B in human liver mitochondria was inhibited noncompetitively, as in human brain synaptosomes. BP‐N‐methylbutylamine was not oxidized by MAO‐A and ‐B. The effects of other BP‐N‐methylalkylamines, such as BP‐N‐methylethylamine, ‐propylamine, and ‐pentanylamine, on MAO activity were examined. BP‐N‐methylbutylamine was the most potent inhibitor of MAO‐A, and BP‐N‐methylethylamine and ‐propylamine inhibited MAO‐B competitively, whereas BP‐N‐methylbutylamine and ‐pentanylamine inhibited it noncompetitively. Inhibition of these BP‐N‐methylalkylamines on MAO‐A and ‐B is discussed in relation to their chemical structure.
Characterization of a High‐Affinity Mg2+‐Independent Ca2+‐ATPase from Rat Brain Synaptosomal Membranes
Authors: Gandhi Chandrashekhar R. , Ross David H.
Published: December 2006

Abstract: A high‐affinity Mg2+‐independent Ca2+‐ATPase (Ca2+‐ATPase) has been differentiated from the Mg2+‐dependent, Ca2+‐stimulated ATPase (Ca2+, Mg2+‐ATPase) in rat brain synaptosomal membranes. Using ATP as a substrate, the K0.5 of Ca2+ for Ca2+‐ATPase was found to be 1.33 μM with a Km for ATP of 19 μM and a Vmax of 33 nmol/mg/min. Using Ca‐ATP as a substrate, the Km for Ca‐ATP was found to be 0.22 μM. Unlike Ca2+, Mg2+‐ATPase, Ca2+‐ATPase was not inhibited by N‐ethylmaleimide, trifluoperazine, lanthanum, zinc, or vanadate. La3+ and Zn2+, in contrast, stimulated the enzyme activity. UnLike Ca2+, Mg2+‐ATPase activity, ATP‐dependent Ca2+ up take was negligible in the absence of added Mg2+, indicating that the Ca2+ transport into synaptosomal endoplasmic reticulum may not be a function of the Ca2+‐ATPase described. Ca2+‐ATPase activity was not stimulated by the monovalent cations Na+ or K+. Ca2+, Mg2+‐ATPase demonstrated a substrate preference for ATP and ADP, but not GTP, whereas Ca2+‐ATPase hydrolyzed ATP and GTP, and to a lesser extent ADP. The results presented here suggest the high‐affinity Mg2+‐independent Ca2+‐ATPase may be a separate form from Ca2+, Mg2+‐ATPase. The capacity of Mg2+‐independent Ca2+‐ATPase to hydrolyze GTP suggests this protein may be involved in GTP‐dependent activities within the cell.
Developmental and Regional Changes of mRNA for a Cerebellar Protein (Spot 35) in the Rat Brain
Authors: Yamakuni Toru , Kuwano Ryozo , Araki Kazuaki , Usui Hiroshi , Inoue Yukichi , Takahashi Yasuo
Published: December 2006

Abstract: Spot 35 protein is a cerebellar Ca‐binding protein. Because Southern blot analysis showed evidence for the nucleotide sequence of spot 35 protein cDNA in the rat genome, we applied cDNA to quantitate the mRNA of rat spot 35 protein. The size of this mRNA was about 1,900 nucleotides in length, and that of mRNA from bovine cerebellum was larger. We also examined the developmental changes and regional distribution of spot 35 protein mRNA in rat brains by dot‐blot analysis using cDNA as a probe. During postnatal days 1–20, a rapid increase of mRNA levels was observed. Further, the level of mRNA for spot 35 protein was found in the cerebellum and was negligible in the cerebral cortex, striatum, and hippocampus.