bluuebunny/crossref_metadata_2025 huggingface dataset:
In Vivo Electrical Stimulation of the Sympathetic Nerve of the Eye Increases Inositol Phosphate Production and Prostaglandin Release in the Rabbit Iris Muscle
Authors: Yousufzai Sardar Y. K. , Gracy Ronald A. , Aboul‐Khair Hisham S. , Abdel‐Latif Ata A.
Published: October 2006

The effects of in vivo electrical stimulation of the sympathetic nerve of the eye on phosphatidylinositol 4,5‐bisphosphate (PIP2) hydrolysis in rabbit iris and release of arachidonate and prostaglandin (PG) E2 into aqueous humor were investigated. myo‐[3H]Inositol or [l‐14C]ara‐chidonate was injected intracamerally into each eye 3 h before electrical stimulation of one of the sympathetic trunks. Tissue phosphoinositides were determined by TLC, and 3H‐Iabeled inositol phosphates were analyzed by either ion‐exchange chromatography or HPLC. The aqueous humor was analyzed for 14C‐labeled arachidonate and PGE2 by radiochromatography and for unlabeled PGE2 by radioimmunoassay. The results obtained from this study can be summarized as follows: (a) The rates of in vivo incorporation of myo‐[3H]inositol into phosphoinositides and accumulation of 3H‐labeled inositol phosphates in the iris muscle increased with time and then leveled off between 3 and 5 h. (b) Distribution of 3H radioactivity in inositol phosphates, as determined by HPLC, showed that of the total radioactivity in inositol phosphates, 53.6% was recovered in myo‐inositol 1‐phosphate. 36% in myo‐inosi‐tol bisphosphate, 0.95% in myo‐inositol 1,3,4‐trisphosphate (1,3,4‐IP3), and 2.6% in 1,4,5‐IP3. (c) Electrical stimulation of the sympathetic nerve resulted in a significant loss of 3H radioactivity from PIP2 and a concomitant increase of that in IP3, an observation indicating that PIP2 is the physiological substrate for α1‐adrenergic receptors in this tissue, (d) Release of IP3 and liberation of arachidonate for PGE2 synthesis are dependent on the duration of stimulation and the intensity (voltage) of stimulus. We conclude that IP3 accumulation and arachidonate release are produced in the iris muscle under in vivo conditions that produce contraction, i.e., electrical stimulation of the sympathetic nerve. These observations add further support to the concept that PIP2 and its derived second messengers are involved in excitation‐contraction coupling in the smooth muscle cells of the iris.
Ionic Requirements for the Specific Binding of [3H]GBR 12783 to a Site Associated with the Dopamine Uptake Carrier
Authors: Bonnet J.‐J. , Benmansour S. , Vaugeois J.‐M. , Costentin J.
Published: October 2006

At 0°C, when Na+ was the only cation present in the incubation medium, increasing the Na+ concentration from 3 to 10 mM enhanced the affinity of [3H]l‐[2‐(di‐phenylmethoxy)ethyl]‐4‐(3‐phenyl‐2‐propenyl)piperazine ([3H]GBR 12783) for the specific binding site present in rat striatal membranes without affecting the 5max. For higher Na+ concentrations, specific binding values plateaued and then slightly decreased at 130 mM Na+. In a 10 mM Na+ medium, the KD and the Bmax were, respectively, 0.23 nM and 12.9 pmol/mg of protein. In the presence of 0.4 nM [3H]GBR 12783, the half‐maximal specific binding occurred at 5 mM Na+. A similar Na+ dependence was observed at 20°C. Scatchard plots indicated that K+, Ca2+, Mg2+, and Tris+ acted like competitive inhibitors of the specific binding of [3H]GBR 12783. The inhibitory potency of various cations (K+, Ca2+, Mg2+, Tris+, Li+ and choline) was enhanced when the Na+ concentration was decreased from 130 to 10 mM. In a 10 mM Na+ medium, the rank order of inhibitory potency was Ca2+ (0.13 mM) > Mg2+ > Tris+ > K+ (15 mM). The requirement for Na+ was rather specific, because none of the other cations acted as a substitute for Na+. No anionic requirement was found: Cl‐, Br‐, and F‐ were equipotent. These results suggest that low Na+ concentrations are required for maximal binding; higher Na+ concentrations protect the specific binding site against the inhibitory effect of other cations.
Phorbol Ester Inhibition of Current Responses and Simultaneous Protein Phosphorylation in Xenopus Oocyte Injected with Brain mRNA
Authors: Kato Ken‐ichi , Kaneko Shuji , Nomura Yasuyuki
Published: October 2006

The role of protein kinase C activation in a coupling of Ca2+‐mobilizing receptors/GTP‐binding protein/ phospholipase C was examined using Xenopus oocytes before and after microinjection of mRNA purified from rat brains. Under voltage‐clamp conditions, although the phorbol ester TPA per se never elicited any changes in ionic conductance, chloride current responses of mRNA‐injected cells to 5‐hydroxytryptamine and acetylcholine (ACh) were suppressed by an 8‐min pretreatment of 12‐O‐tetradeca‐noyl‐4β‐phorbol‐13‐acetate (TPA), at nanomolar concentrations. Native ACh response in intact follicular oocytes was also inhibited by the TPA treatment. However, similar current responses triggered by the direct activation of their intracellular signalling pathway with guanosine‐5′‐O‐(3‐thio)triphosphate or Ca2+ were not affected by TPA. Biochemical analyses indicated that phosphorylation of 33,000‐ and 45,000‐dalton proteins was markedly enhanced by TPA in vivo, and that stimulation of receptors with agonists as well as TPA treatment increased phospho‐proteins in the membrane fraction of mRNA‐injected oocytes. These observations suggest that protein kinase C may switch off the signal transduction from receptors to GTP‐binding proteins and may participate in the negative feedback modulation of receptor‐operated ion channel responses.
Acylgalactosylceramides in Developing Dysmyelinating Mutant Mice
Authors: Theret N. , Boulenguer P. , Fournet B. , Fruchart J. C. , Bourre J. M. , Delbart C.
Published: October 2006

Acylgalactosylceramides (AGC) from forebrains of normal and dysmyelinating (quaking and shiverer) mice were purified by Florisil column chromatography and preparative TLC. These procedures resolved the AGC on the basis of their Rf values into two main fractions which co‐nigrate with their homologs from rat forebrains. In control animals, AGC were detectable in mouse forebrains from the eighth postnatal day and reached maximal values within 20 days. The same developmental pattern was obtained in dysmyelinating shiverer mice but the AGC content was reduced to approximately 30% of control values. In quaking mutants, the AGC were hardly detected. They were also present in sciatic nerve of normal mice and to a lesser extent in trembler mice. Gas chromatography‐mass spectrometry analysis of both ester‐ and amide‐linked fatty acids isolated from AGC of normal and shiverer mice shows that the shiverer mutant AGC display a chemical structure similar to that of normal AGC. AGC constituents of control myelin are reduced by approximately 70% in shiverer myelin, indicating that these molecules can be considered as early markers of oligodendrocyte differentiation. The early arrest of myelinogenesis in the quaking animals and the near absence of AGC are in good agreement with this proposal. Moreover, the reduced amount of AGC in the trembler PNS indicates that AGC could also be early markers for differentiation of the Schwann cell.
Interaction of l‐Prolyl‐l‐Leucyl Glycinamide with Dopamine D2 Receptor: Evidence for Modulation of Agonist Affinity States in Bovine Striatal Membranes
Authors: Srivastava Lalit K. , Bajwa Samina B. , Johnson Rodney L. , Mishra Ram K.
Published: October 2006

The role of the hypothalamic tripeptide l‐prolyl‐l‐leucyl‐glycinamide (PLG) in modulating the agonist binding to bovine striatal dopamine D2 receptor was investigated using a selective high‐affinity agonist, n‐propylnor‐apomorphine (NPA). PLG caused an enhancement in [3H]NPA binding in striatal membranes in a dose‐dependent manner, the maximum effect being observed at 10‐‐7‐‐10‐‐6M concentration of the tripeptide. The Scat‐chard analysis of [3H]NPA binding to membranes preincu‐bated with 10‐‐6M PLG revealed a significant increase in the affinity of the agonist binding sites. In contrast, there was no effect of PLG on the binding pattern of the antagonist [3H]spiroperidol. The antagonist versus agonist competition curves analyzed for agonist high‐ and low‐affinity states of the receptor displayed an increase in the population and affinity of the high‐affinity form of the receptor with PLG treatment. The low‐affinity sites concomitantly decreased with relatively small change in the affinity for the agonists. Almost similar results were obtained when either NPA or apomorphine was used in the competition experiments. A partial antagonistic effect of PLG on 5′‐guanylyl‐imidodiphosphate [Gpp(NH)p]‐induced inhibition of high‐affinity agonist binding was also observed, as the ratio of high‐ to low‐affinity forms of the receptor was significantly higher in the PLG‐treated membranes compared to the controls. Direct [3H]NPA binding experiments demonstrated that PLG attenuated the Gpp(NH)p‐induced inhibition of agonist binding by increasing the EC50 of the nu‐cleotide (concentration that inhibits 50% of the specific binding). No effect of PLG on high‐affinity [3H]NPA binding, however, could be observed when the striatal membranes were preincubated with Gpp(NH)p. The binding of antagonists and agonists to α2 adrenergic receptors, negatively coupled to adenylate cyclase in the striatum. was not affected by PLG. The results suggest that PLG modulates the affinity states of the dopamine D2 receptor, possibly by enhancing its interaction with the guanine nucleotide regulatory protein.
Altered Cerebral Protein Turnover in Rats Following Prolonged In Vivo Treatment with Nicotine
Authors: Katyare Surendra S. , Shallom Joshua M.
Published: October 2006

Abstract: Turnover rates of cerebral proteins were examined in control adult rats and in those subjected to prolonged in vivo treatment with “low” (0.02 mg/ml) or “high” (0.04 mg/ml) doses of nicotine (added to drinking water), using [14C]bicarbonate as the label. It was found that the turnover of proteins in various subcellular fractions consisted of two distinct components turning over at a “fast” or a “slow” rate and having relatively short or long half‐lives, respectively. Thus in control animals the half‐lives of the protein components turning over at a fast rate ranged from 1.31 to 3.61 days whereas for those turning over at a slow rate the half‐lives ranged from 8.56 to 24.28 days. Treatment with low doses of nicotine resulted in a more rapid turnover of nuclear fast turning over component with a concomitant decreased turnover of homogenate, cytosol, mitochondrial, and microsomal proteins; in the synaptosomal membranes this component disappeared altogether. The half‐lives of the slow turning over components decreased in general from 14.3 to 33.3% with the exception of the nuclear proteins, where the half‐life increased by 71.1%. Turnover of microsomal proteins was not affected. When the animals were given a high dose of nicotine, the turnover of fast components became even more rapid for nuclear, myelin, and microsomal proteins with a decrease in half‐life from 26.6 to 32.3%. By contrast, half‐lives of synaptosomal and mitochondrial proteins increased by 16.1–89.3%. These changes were not reflected in the turnover rate of whole homogenate proteins. Turnover rates of even the slow components increased for cytosol, nuclear, homogenate, and myelin proteins with decrease in t1/2 from 22.9 to 60.2%. By contrast, t1/2 of microsomal and mitochondrial proteins increased by 51.1 and 66.4%. Turnover of synaptosomal proteins was not affected under these conditions. Treatment with nicotine also brought about a small but reproducible increase in the rate of turnover of liver homogenate proteins which is attributable almost completely to the nuclear proteins. With low doses of nicotine half‐life of mitochondrial proteins increased by 7.2% whereas that of microsomal proteins decreased by 8.5%. The results thus emphasize that in vivo effects of nicotine are more specific for cerebral protein metabolism.
Long‐Term Changes in Rat Brain Tyrosine Hydroxylase Following Reserpine Treatment: A Quantitative Immunochemical Analysis
Authors: Labatut Rene , Buda Michel , Berod Anne
Published: October 2006

Abstract: An immunoblot procedure was developed to quantify the amount of tyrosine hydroxylase protein in homogenate of small brain regions. With the use of this method we have studied the variations in tyrosine hydroxylase activity and protein levels in some catecholaminergic neurons at different times following a single reserpine injection (10 mg/kg s.c.) and reevaluated the anatomical specificity of tyrosine hydroxylase induction by this drug. Reserpine administration provoked a long‐lasting increase in both tyrosine hydroxylase activity and protein levels within locus ceruleus neurons. This effect culminated at day 4 after injection. At this time, the enzyme activity and protein levels in treated animals were respectively 2.7 and 2.6 times that measured in vehicle‐treated animals. Both parameters varied in parallel so that tyrosine hydroxylase specific activity did not change over time. In contrast, reserpine did not cause any changes in tyrosine hydroxylase activity in the dopaminergic neurons of the substantia nigra, but provoked a moderate increase in tyrosine hydroxylase protein level. This latter effect was maximal (1.5 times) 4 days after treatment. In the adjacent dopaminergic area, i.e., the ventral tegmental area, a small decrease in the enzyme activity was recorded at day 2 without any significant change in the level of the protein. In conclusion, first, our data show the capacity of our method to assay tyrosine hydroxylase protein amounts in small brain catecholaminergic nuclei. Second, our results confirm and extend previous studies on the effect of reserpine on the regulation of tyrosine hydroxylase level within brain noradrenergic neurons. They further show that within the dopaminergic neurons of the substantia nigra reserpine administration can alter tyrosine hydroxylase protein levels without changes in its activity.
Voltage‐Sensitive Calcium Channels in Differentiated Neuroblastoma × Glioma Hybrid (NG108–15) Cells: Characterization by Quin 2 Fluorescence
Authors: Noronha‐Blob L. , Richard C. , U'Prichard D. C.
Published: October 2006

Abstract: Depolarization of differentiated neuroblastoma × glioma (NG108–15) cells with KC1 (50 mM) or veratridine (50 μM) stimulated Ca2+ accumulation, was detected by quin 2 fluorescence. Intracellular Ca2+ concentrations ([Ca2+]i) were elevated about threefold from 159 ± 7 to 595 ± 52 nM (n = 12). Ca2+ entry evoked by high extracellular K+ concentration ([K+]o) was voltage‐dependent and enhanced by the dihydropyridine agonists, BAY K 8644 and CGP 28 392, in a dose‐dependent manner. CGP 28 392 was less potent and less efficacious than BAY K 8644. The (+) and (‐) stereoisomers of 202–791 showed agonist and antagonist properties, respectively. (+)‐202–791 was less potent, but as efficacious as BAY K 8644. In the absence of KCl, BAY K 8644 had no effect on Ca2+ entry. Voltage‐sensitive calcium channel (VSCC) activity was blocked by organic Ca2+ channel antagonists (nanomolar range) both before and after KC1 treatment and also by divalent metal cations (micromolar range). High [K+]o‐in‐duced Ca2+ accumulation was dependent on external Ca2+, but not on external Na+ ions ([Na]o), and was insensitive to both tetrodotoxin (3 μM) and tetraethylammonium (10 μM). In contrast, veratridine‐induced Ca2+ accumulation required [Na+]o, and was blocked by tetrodotoxin, but not by nimodipine (1 μM). Veratridine‐induced Ca2+ accumulation was slower (∼45 s), smaller in magnitude (∼30% of [K+]o‐induced Ca2+ entry), and also enhanced by BAY K 8644 (∼50%). VSCC were identified in neuronal hybrid (NG108–15 and NCB‐20) cells, but not in glial (C6BU‐1), renal epithelial (MDCK), and human astrocytoma (1321N1) cells. NG108–15 cells differentiated with 1.0 mM dibutyryl cyclic AMP showed greater VSCC activity than undifferentiated cultures. These results suggest that cultured neural cells provide a useful system to study Ca2+ regulation via ion channels.
Ketone Body Utilization for Lipid Synthesis in the Murine Sciatic Nerve: Alterations in the Dysmyelinating Trembler Mutant
Authors: Clouet Perrine M. , Bourre Jean‐Marie
Published: October 2006

Abstract: This work demonstrates that in vitro sciatic nerves of normal and trembler adult mice can use ketone bodies (β‐hydroxybutyrate and acetoacetate) and butyrate for lipid synthesis. In normal sciatic nerves, β‐hydroxybutyrate is incorporated in total lipids to a larger extent than acetoacetate (141% and 33%, respectively, of acetate incorporation), whereas for trembler sciatic nerves, these percentages are only 69% and 27%. Incorporation of ketone bodies is greater into sterols than into other lipids. Lipid metabolism of ketone bodies in trembler nerves is altered and could reflect a process similar to Wallerian degeneration: a dramatic decrease of sterol and free fatty acid synthesis and an increased synthesis of triglycerides. Moreover, differences seen in precursor incorporation into lipids between normal and trembler sciatic nerves suggest that their lipid metabolism is not the same.
Contents of Cystathionine and Taurine in Various Cerebellar Regions of dl‐Propargylglycine‐Treated Rats
Authors: Kodama Hiroyuki , Sasaki Keiko , Mizobuchi Noriko , Kikuchi Reiji
Published: October 2006

Abstract: The contents of cystathionine and taurine, as well as cystathionine β‐synthase activity, in various cerebellar regions and pineal body of normal and dl‐propargylgly‐cine‐treated rats were measured. The contents of cystathionine and taurine were found to be distributed unevenly in cerebellar regions of brain of both normal and dl‐propargylglycine‐treated rats. The content of cystathionine in each cerebellar region and pineal body increased gradually when the dose of DL‐propargylglycine was increased from 10 mg to 30 mg per 200 g body weight. On the other hand, taurine content in each cerebellar region and pineal body decreased with the administration of 30 mg of dl‐propargylglycine per 200 g body weight. The contents of cystathionine and taurine were greater in the pineal body than in various cerebellar regions. The activity of cystathionine β‐synthase was also distributed unevenly in various cerebellar regions of normal rat brain, and was unaltered following treatment of rats with dl‐propargylglycine.
Anion Regulation of Agonist and Inverse Agonist Binding to Benzodiazepine Receptors
Authors: Evoniuk Gary , Skolnick Phil
Published: October 2006

Abstract: Binding of the benzodiazepine inverse agonist [3H]methyl‐6,7‐dimethoxy‐4‐ethyl‐β‐carboline‐3‐carboxylate ([3H]DMCM) and the agonist [3H]flunitrazepam ([3H]FNZ) was compared in rat cortical membranes. Halide ions enhanced [3H]DMCM binding three‐ to fourfold, increasing both the apparent affinity and the number of binding sites for this radioligand. The effect was present at both 0 and 37°C. In contrast, the magnitude of halide stimulation of [3H]FNZ binding was much smaller, resulting solely from an increase in the apparent affinity for this radioligand, and was not observed at 37°C. The potencies but not the efficacies of a series of anions to stimulate both [3H]DMCM and [3H]FNZ binding to benzodiazepine receptors were highly correlated with their relative permeabilities through γ‐aminobutyric acid (GABA)‐gated chloride channels. Two stress paradigms (10 min of immobilization or ambient‐temperature swim stress), previously shown to increase significantly the magnitude of halide‐stimulated [3H]FNZ binding, did not significantly affect [3H]DMCM binding. Phospholipase A2 treatment of cortical membrane preparations was equipotent in preventing the stimulatory effect of chloride on both [3H]DMCM and [3H]FNZ binding. These data strongly suggest that anions modify the binding of [3H]DMCM and [3H]FNZ by acting at a common anion binding site that is an integral component of the GABA/benzodiazepine receptor chloride channel complex.
Inactivation of Tyrosine Hydroxylase Activity by Ascorbate In Vitro and in Rat PC12 Cells
Authors: Wilgus Harvey , Roskoski Robert
Published: October 2006

Abstract: Tyrosine hydroxylase activity is reversibly modulated by the actions of a number of protein kinases and phosphoprotein phosphatases. A previous report from this laboratory showed that low‐molecular‐weight substances present in striatal extracts lead to an irreversible loss of tyrosine hydroxylase activity under cyclic AMP‐dependent phosphorylation conditions. We report here that ascorbate is one agent that inactivates striatal tyrosine hydroxylase activity with an EC50 of 5.9 μM under phosphorylating conditions. Much higher concentrations (100 mM) fail to inactivate the enzyme under nonphosphorylating conditions. Isoascorbate (EC50, 11 μM) and dehydroascorbate (EC50, 970 μM) also inactivated tyrosine hydroxylase under phosphorylating but not under nonphosphorylating conditions. In contrast, ascorbate sulfate was inactive under phosphorylating conditions at concentrations up to 100 mM. Since the reduced compounds generate several reactive species in the presence of oxygen, the possible protecting effects of catalase, peroxidase, and superoxide dismutase were examined. None of these three enzymes, however, afforded any protection against inactivation. We also examined the effects of ascorbate and its congeners on the activity of tyrosine hydroxylase purified to near homogeneity from a rat pheochromocytoma. This purified enzyme was also inactivated by the same agents that inactivated the impure corpus striatal enzyme. Under conditions in which ascorbate almost completely abolished enzyme activity, we found no indication for significant prote‐olysis of the purified enzyme as determined by sodium do‐decyl sulfate‐polyacrylamide gel electrophoresis. We also found that pretreatment of PC12 cells in culture for 4 h with 1 mM ascorbate, dehydroascorbate, or isoascorbate (but not ascorbate sulfate) also decreased tyrosine hydroxylase activity 25–50%. The inactivation seen under in vitro conditions appears to have a counterpart under more physiological conditions.
Rapid Increases in Glial Fibrillary Acidic Protein mRNA and Protein Levels in the Copper‐Deficient, Brindled Mouse
Authors: Shafit‐Zagardo Bridget , Peterson C. , Goldman James E.
Published: October 2006

Abstract: The brindled mouse (Mobr/y) carries an X‐linked mutation that produces severe copper deficiency. Affected males suffer profound deficits in oxidative metabolism. We have examined astrocyte pathology in Mobr/y during development and have found marked changes in the metabolism of glial fibrillary acidic protein (GFAP). Immunocytochemistry with anti‐GFAP antisera revealed a marked increase in staining at postnatal day 12 (P12), compared to heterozygous female and unaffected male littermates, particularly in neocortex and thalamus. Septum, hypothalamus, and striatum showed little change. Western blot analysis revealed increased levels of GFAP in Mobr/y forebrain and cerebellum. Levels of GFAP mRNA were determined by Northern blotting with a mouse GFAP cDNA probe. At P10, mRNA levels were normal, but increased to 8–10 times normal by P12. Levels at P15 remained similarly elevated. Thus, immunostaining and protein determinations correlate with mRNA elevations. Astrocytes can alter GFAP mRNA and protein levels over a relatively short time. Counts of neocortical cells did not reveal differences in cell numbers between Mobr/y and controls, indicating that the observed changes reflect increased cellular levels and not a large increase in the numbers of astrocytes.
Importance of Glutamine for γ‐Aminobutyric Acid Synthesis in Rat Neostriatum In Vivo
Authors: Paulsen R. E. , Odden E. , Fonnum F.
Published: October 2006

Abstract: This work was carried out to evaluate the importance of glial cells in providing precursors for the in vivo synthesis of γ‐aminobutyric acid (GABA). Fluorocitrate, which selectively inhibits the tricarboxylic acid cycle in glial cells, was administered locally in rat neostriatum. Inhibition of the glial cell tricarboxylic acid cycle led to a decrease both in glutamine level and in γ‐vinyl GABA (GVG)‐induced GABA accumulation, an observation indicating reduced GABA synthesis. The role of glutamine, which is synthesized in glial cells as a precursor for GABA, was further investigated by inhibition of glutamine synthetase with intrastriatally administered methionine sulfoximine. In this case, the glutamine level was reduced to near zero values, and the GVG‐induced GABA accumulation was only half that of normal. The results show that glutamine is an important precursor for GABA synthesis, but it cannot be the sole precursor because it was not possible to depress the GVG‐induced GABA accumulation completely.
Structural Features of Human Monoamine Oxidase A Elucidated from cDNA and Peptide Sequences
Authors: Hsu Yun‐Pung P. , Weyler Walter , Chen Shiuan , Sims Katherine B. , Rinehart William B. , Utterback Margot C. , Powell John F. , Breakefield Xandra O.
Published: October 2006

Abstract: Monoamine oxidase (MAO), an important enzyme for the degradation of amine neurotransmitters, has been implicated in neu‐ropsychiatric illness. The amino acid sequence for one form of the enzyme, MAO‐A, has been deduced from human cDNA clones and verified against proteolytic peptides. The covalent binding site for the flavin adenine dinucleotide (FAD) cofactor is near the C‐terminal region. The presence of features characteristic of the ADP‐binding fold suggests that the N‐terminal region is also involved in the binding of FAD. These cDNAs should facilitate the study of the structure, function, and intracellular targeting of MAO, as well as the analysis of its expression in normal and pathological states.
Age‐Related Changes of Ribonuclease Activities in Various Regions of the Rat Central Nervous System
Authors: Alberghina M. , Stella A. M. Giuffrida
Published: October 2006

Acid (pH 5.5), free, and latent alkaline (pH 7.4) RNases were assayed in homogenates of temporal cortex, hypothalamus, hippocampus, and cervicothoracic segments of spinal cord of rats at three different ages (5,14, and 25 months old). Free alkaline RNase activity was lower (two‐ to fivefold) than the acid activity. Both free and inhibitor‐bound alkaline RNases remained unchanged with age in all CNS regions examined. This result also indirectly indicates no change of RNase‐inhibitor complex throughout aging. In contrast, the acid RNase activity showed a significant increase during aging in all tissues, with exception of the hypothalamus. Because this enzyme is localized mainly in the lysosomes, this result might be due to an increased lysosomal activity and/or to the release of hydro‐lases into the cytoplasm from these organelles, undergoing shrinkage and degeneration in aged animals.
Bicuculline Up‐Regulation of GABAA Receptors in Rat Brain
Authors: Ito Yoshihisa , Lim Dong Koo , Hoskins Beth , Ho Ing K.
Published: October 2006

Effects of acute and subacute administration of Dicuculline on [3H]muscimol, [3H]flunitrazepam, and t‐[35S]butylbicyclophosphorothionate ([35S]TBPS) binding to various brain regions were studied in Sprague‐Dawley rats. Acute administration of bicuculline affected neither the KD nor the Bmax of the three receptor sites. In rats treated sub‐acutely with bicuculline (2 mg/kg, i.p., daily for 10 days), [3H]muscimol binding was increased in the frontal cortex, cerebellum, striatum, and substantia nigra. Scatchard analysis revealed that subacute treatment of rats with bicuculline resulted in a significantly lower KD of high‐affinity sites in the striatum and in a significantly lower KD of high‐ and low‐affinity sites in the frontal cortex. In the cerebellum, two binding sites were apparent in controls and acutely treated animals; however, only the high‐affinity site was defined in subacutely treated animals, with an increase in the Bmax value. Triton X‐100 treatment of frontal cortical membranes eliminated the difference in [3H]muscimol binding between control and subacute bicuculline treatments. On the other hand, [3H]muscimol binding was significantly increased in the cerebellum from bicuculline‐treated animals even after Triton X‐100 treatment. The apparent Ki of bicuculline for the GABAA receptor was also decreased in the frontal cortex and the striatum following the treatment. However, subacute administration of bicuculline affected neither the KD nor the Bmax of [3H]‐flunitrazepam and [35S]TBPS binding in the frontal cortex and the cerebellum. These results suggest that GABAA receptors are up‐regulated after subacute administration of bicuculline, with no change in benzodiazepine and picro‐toxin binding sites.
Exchange of Guanine Nucleotides Between Tubulin and GTP‐Binding Proteins That Regulate Adenylate Cyclase: Cytoskeletal Modification of Neuronal Signal Transduction
Authors: Rasenick Mark M. , Wang Nan
Published: October 2006

Tubulin, the primary constituent of microtubules, is a GTP‐binding protein with structural similarities to other GTP‐binding proteins. Whereas microtubules have been implicated as modulators of the adenylate cyclase system, the mechanism of this regulation has been elusive. Tubulin, polymerized with the hydrolysis‐resistant GTP analog, 5′‐guanylylimidodiphosphate [Gpp(NH)p], can promote inhibition of synaptic membrane adenylate cyclase which persists subsequent to washing. Tubulin with Gpp(NH)p bound was slightly less potent than free Gpp(NH)p in the inhibition of adenylate cyclase, but tubulin without nucleotide bound had no effect on the enzyme. A GTP‐binding protein from the rod outer segment (transducin), with Gpp(NH)p bound, was also without effect on adenylate cyclase. Tubulin (regardless of the nucleotide bound to it) did not alter the activity of the adenylate cyclase catalytic unit directly. When tubulin was polymerized with the hydrolysis‐resistant photoaffinity GTP analog, [32P]P3(4‐azidoanilido)‐P1‐5′‐GTP ([32P]AAGTP), and this protein was added to synaptic membranes, AAGTP was transferred from tubulin to the inhibitory GTP‐binding protein, Gi. This transfer was blocked by prior incubation of the membranes with Gpp(NH)p or covalent binding of AAGTP to tubulin prior to exposure of that tubulin to membranes. Incubation of membranes with Gpp(NH)p subsequent to incubation with tubulin‐AAGTP results in a decrease in AAGTP bound to Gi and a compensatory increase in AAGTP bound to the stimulatory GTP‐binding protein, Gs. Likewise, persistent inhibition of adenylate cyclase by tubulin‐Gpp(NH)p could be overridden by the inclusion of 100 μM Gpp(NH)p in the assay inhibition. Whereas Gpp(NH)p promotes persistent inhibition of synaptic membrane adenylate cyclase without incubation at elevated temperatures, tubulin [with AAGTP or Gpp(NH)p bound] requires 30 s incubation at 23°C to effect adenylate cyclase inhibition. Photoaffinity experiments yield parallel results. These data are consistent with synaptic membrane tubulin regulating neuronal adenylate cyclase by transferring GTP to Gi and, subsequently, to Gs.
Demonstration and Biochemical Characterisation of Rat Brain NADPH‐Dependent Diaphorase
Authors: Kuonen Donald R. , Kemp Martyn C. , Roberts Peter J.
Published: October 2006

Abstract: An enzyme responsible for the NADPH‐dependent reduction of nitroblue tetrazolium HCl (NBT) has been isolated from rat brain. Although other tetrazolium salts could be utilised, NBT was the preferred substrate, and the enzyme had an absolute requirement for NADPH. An in vitro assay was developed and used to determine the kinetic constants: Km NBT = 17.3 μM: Km NADPH = 1.9 μM, Vmax= 30.8 μmol product produced/min/mg protein. Substrate inhibition by NADPH was observed in some instances. Brain subcellular fractionation indicated highest enzyme activities in the microsomal fraction. Activity was present in all brain regions and in a variety of peripheral tissues. Relative molecular mass determinations of the native enzyme yielded an Mr= 170–180.000. It seems likely that the enzyme activity described in this study relates directly to the histochemical demonstration of brain NADPH‐diaphorase‐positive neurons. As yet, the natural substrate for the enzyme is unknown. However, the isolation and purification of NADPH‐dependent diaphorase may be anticipated to assist in the elucidation of its function in the brain, and in the special characteristics of those neurons that contain the enzyme in abundance.
[3H]Imipramine Binding of Protein Nature in Human Platelets: Inhibition by 5‐Hydroxytryptamine and 5‐Hydroxytryptamine Uptake Inhibitors
Authors: Marcusson Jan , Tiger Gunnar
Published: October 2006

Abstract: The nature of [3H]imipramine binding to human platelets was investigated. Desipramine and 5‐hydroxytryptamine (5‐HT) displaced the same amount of binding and the binding was sensitive to protease treatment. The nature of pharmacological inhibition of [3H]imipramine binding was investigated in saturation experiments. Increases in Kd without changes in Bmax were noted with the addition of 5‐HT, desipramine, norzimeldine, or 5‐methoxytryptoline. Reductions in Bmax without alterations in KD were obtained when citalopram or clomipramine was added. It is concluded that the [3H]imipramine binding site in human platelets is of protein nature and that this binding site contains the substrate recognition site for 5‐HT uptake. In addition, [3H]imipramine and other 5‐HT uptake inhibitors have bonds to other parts of the 5‐HT uptake carrier or to the surrounding lipid membrane. This additional binding outside the substrate recognition site is not one single site but most likely represents sites that are specific for the chemical structure of each uptake inhibitor, respectively.
Myelin Basic Protein Is an Endogenous Inhibitor of the High‐Affinity Cannabinoid Binding Site in Brain
Authors: Nye Jeffrey S. , Voglmaier Susan , Martenson Russell E. , Snyder Solomon H.
Published: October 2006

Abstract: Radioligand binding studies with the water‐soluble cannabinoid [3H]5′‐trimethylammonium A8‐tetrahydrocannabinol ([3H]TMA) have revealed a saturable highaffinity site in brain that is specific for cannabinoids. To determine whether endogenous compounds of brain might act upon the site physiologically, we sought inhibitors in extracts of brain. An endogenous inhibitor has been purified to homogeneity by acid extraction of rat brain followed by adsorption to a reverse‐phase matrix and gel filtration chromatography. The purified inhibitor has a subunit molecular mass of 14,500 daltons by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS‐PAGE). Inhibition of [3H]TMA binding by the purified inhibitor occurs with a Ki of about 4 nM in a noncompetitive manner. The molecular weight, abundance, and extraction properties are the same as a species of myelin basic protein (MBP). The MBPs of rat, rabbit, pig, and cow also inhibit [3H]TMA binding noncompetitively with similar potencies. The purified inhibitor comigrates with rat MBP‐small form on SDS‐PAGE, has a similar amino acid composition, and is recognized by antibody directed against MBP. Studies of fragments of rabbit MBP suggest that the determinants of affinity for the [3H]TMA site are contained primarily within the C‐terminal half of the rabbit MBP. Synthetic polycationic peptides such as polylysine and polyarginine mimic the effects of MBP, suggesting that the high‐affinity cannabinoid binding site recognizes large polycations. The identification of the endogenous inhibitor of [3H]TMA binding as MBP suggests that MBP interacts physiologically with the high‐affinity cannabinoid site.
Histamine Metabolites in Cerebrospinal Fluid of the Rhesus Monkey (Macaca mulatto): Cisternal‐Lumbar Concentration Gradients
Authors: Prell George D. , Khandelwal Jai K. , Burns R. Stanley , Green Jack Peter
Published: October 2006

Abstract: Similar to metabolites of other aminergic transmitters, histamine metabolites of brain, tele‐methylhistamine (t‐MH) and tele‐methylimidazoleacetic acid (t‐MIAA), could have a concentration gradient between rostral and caudal sites of CSF. To test this hypothesis, cisternal and lumbar CSF samples were collected in pairs from eight monkeys (Macaca mulatta), and levels of t‐MH and t‐MIAA were measured by gas chromatography‐mass spectrometry. pros‐Methylimidazoleacetic acid (p‐MIAA), an endogenous isomer of t‐MIAA that is not a histamine metabolite, was also measured. Cisternal levels (in picomoles per milliliter, mean ± SEM) of t‐MH (9.9 ± 1.4) and t‐MIAA (40.8 ± 7.6), but not of p‐MIAA (9.7 ± 1.2), exceeded those in lumbar CSF (t‐MH, 1.8 ± 0.3; t‐MIAA, 6.8 ± 0.9; p‐MIAA, 8.6 ± 0.6) in every monkey. The magnitudes of the mean cisternal‐lumbar concentration gradients fort‐MH(6.6 ± 1.1)and t‐MIAA (6.5 ± 1.3) were indistinguishable. These gradients exceed those of metabolites of most other transmitters. There was no gradient for the levels of p‐MIAA. The cisternal, but not lumbar, levels of t‐MH and t‐MIAA were correlated. There was no significant difference between the means of the metabolite concentration ratios (t‐MIAAJ.t‐MH) in cisternal (4.0 ± 0.4) and lumbar (4.4 ± 0.9) CSF. The steepness of these gradients suggests that levels of t‐MH and t‐MIAA in lumbar CSF might be useful probes of histaminergic metabolism in brain.
Hydrolysis of the Brain Dipeptide N‐Acetyl‐l‐Aspartyl‐l‐Glutamate: Subcellular and Regional Distribution, Ontogeny, and the Effect of Lesions on N‐Acetylated‐α‐Linked Acidic Dipeptidase Activity
Authors: Blakely Randy D. , Robinson Michael B. , Thompson Reid C. , Coyle Joseph T.
Published: October 2006

Abstract: N‐Acetylated‐α‐linked acidic dipeptidase (NAA‐LADase) is a Cl− dependent, membrane bound, metallopeptidase that cleaves the endogenous neuropeptide N‐acetyl‐l‐aspartyl‐l‐glutamate (NAAG) in vitro. To examine the pattern of NAALADase expression in the CNS, subcellular, regional, and developmental studies were conducted. Subcellular fractionation of lysed synaptosomal membranes revealed a substantial enrichment of the peptidase in synaptic plasma membranes as compared to mitochondrial or myelin subfractions. Regional studies reveal an apparent restriction of peptidase activity to kidney and brain. A threefold variation in specific activity was observed among brain regions, with highest specific activity in the cerebellum and lowest in telencephalic structures, a pattern that does not, in general, correlate with NAAG levels. Ontogenetic studies demonstrate a region‐dependent, postnatal pattern of expression of NAALADase activity, with adult levels attained earliest in brainstem, as was previously reported for NAAG. Postnatal NAALADase expression would not appear to support a role for the peptidase in constitutive protein processing, but rather suggests that NAALADase may play a role in synaptic peptide degradation. Glutamate (Glu) excised from NAAG by NAALA Dase could be transported efficiently by uptake processes. Lesion studies, however, do not support a close structural association between NAALADase activity and the corticostriatal sodium‐dependent, high‐affinity, Glu uptake system. Similar to in vitro data documenting the route of NAAG degradation by NAALADase, after intrastriatal injection, NAAG was rapidly cleaved to two major products. N‐acetyl‐aspartate and Glu, with a t1/2 of approximately 10 min. Thus, the route of in vivo catabolism of NAAG parallels results from studies on NAALADase activity in vitro. These results are consistent with a role of NAALADase in the synaptic processing of NAAG. However, certain discrepancies in the regional and ontogenetic profiles of NAAG and NAALADase suggest that this relationship is not an exclusive one and may reflect a role for NAALADase on additional N‐acetylated acidic peptides in vivo.
Effect of Pyrithiamine Treatment and Subsequent Thiamine Rehabilitation on Regional Cerebral Amino Acids and Thiamine‐Dependent Enzymes
Authors: Butterworth Roger F. , Héroux Maryse
Published: October 2006

Abstract Pyrithiamine‐induced thiamine‐deficiency encephalopathy in the rat shows many neuropathologies and biochemical similarities to Wernicke's encephalopathy in humans. Treatment of rats with pyrithiamine resulted in moderate reductions of glutamate in thalamus and pons and in generalized severe reductions of aspartate in pons (by 89%, p < 0.01), thalamus (by 83%, p < 0.01), cerebellum (by 53%, p < 0.01), and cerebral cortex (by 33%, p < 0.05). Alanine concentrations were concomitantly increased. Activites of the thiamine‐dependent enzyme α‐ketoglutarate dehydrogenase (αKGDH) were decreased in parallel with the aspartate decreases; pyruvate dehydrogenase complex activities were unchanged in all brain regions. Following thiamine administration to symptomatic pyrithiamine‐treated rats, neurological symptoms were reversed and concentrations of glutamate, aspartate, and alanine, as well as αKGDH activities, were restored to normal in cerebral cortex and pons. Aspartate levels and αKGDH activities remained below normal values, however, in thalamus. Thus, pyrithiamine treatment leads to reductions of cerebral αKGDH and (1) decreased glucose (pyruvate) oxidation resulting in accumulation of alanine and (2) decreased brain content of glutamate and aspartate. Such changes may be of key significance in the pathophysiology of the reversible and irreversible signs of Wernicke's encephalopathy in humans.
Synthesis and Release of Dopamine in Rat Brain: Comparison Between Substantia Nigra Pars Compacta, Pars Reticulata, and Striatum
Authors: Nissbrandt H. , Sundström E , Jonsson G , Hjorth S. , Carlsson A.
Published: October 2006

Abstract Dopamine (DA) is synthesized and released not only from the terminals of the nigrostriatal dopaminergic neuronal pathway, but also from the dendrites in the substantia nigra. We have investigated the regulation of the DA turnover, the DA synthesis rate, and the DA release in the substantia nigra pars compacta (SNpc) and pars reticulata (SNpr) in vivo. As a measure of DA turnover, we have assessed the concentrations of 3,4‐dihydroxyphenylacetic acid and homovanillic acid. As a measure of the DA synthesis rate, we have determined the 3,4‐dihydroxyphenylalanine accumulation after inhibition of aromatic l‐amino acid decarboxylase by 3‐hydroxybenzylhydrazine. As a measure of DA release, we have investigated the disappearance rate of DA after inhibition of its synthesis by α‐methyl‐p‐tyrosine and the 3‐methoxytyramine accumulation following monoamine oxidase inhibition by pargyline. Both the DA turnover and the DA synthesis rate increased following treatment with the DA receptor antagonist haloperidol and decreased following treatment with the DA receptor agonist apomoiphine in the SNpc and in the SNpr, but the effects of the drugs were less pronounced than in the striatum. γ‐Butyrolactone treatment, which suppresses the firing of the dopaminergic neurons, increased the DA synthesis rate in the striatum (165%), but had no such effect in the SNpc or SNpr. Haloperidol, apomorphine, and γ‐butyrolactone increased, decreased, and abolished, respectively, the DA release in the striatum, but the drugs had no or only slight effects on the α‐methyl‐p‐tyrosine‐induced DA disappearance and on the pargyline‐induced 3‐methoxytyramine accumulation in the SNpc or SNpr. Taken together, these results indicate that the DA synthesis rate, but not the DA release, are influenced by DA receptor activity and neuronal firing in the SNpc and SNpr. This is in contrast to the situation in the striatum, where both the DA synthesis rate and the DA release are under such control.
Octanoic Acid Inhibits Astrocyte Volume Control: Implications for Cerebral Edema in Reye's Syndrome
Authors: Olson James E. , Holtzman David , Sankar Raman , Lawson Christyne , Rosenberg Robert
Published: October 2006

Abstract Octanoic acid has been implicated in the pathogenesis of cytotoxic cerebral edema in Reye's syndrome. Using astrocytes from primary culture, we studied the dose‐dependent effects of octanoate on cellular volume regulation and metabolism. Astrocyte volume recovery following hypoosmotic swelling was stimulated by 1.0 mM octanoate and inhibited by 3.0 mM octanoate. Parallel effects were obtained at these concentrations on the activity of the Na+,K+‐depen‐dent ATPase. Cellular ATP concentrations also were reduced 36% with the higher octanoate concentration. These effects of octanoate may contribute to the severe astrocyte swelling observed in the brains of Reye's syndrome patients.
Protein Phosphorylation Induced by Phorbol Esters and Cyclic AMP in Anterior Pituitary Cells: Possible Role in Adrenocorticotropin Release and Synthesis
Authors: Rougon Genevieve , Barbet Jacques , Reisine Terry
Published: October 2006

Abstract: Forskolin, an activator of adenylate cyclase, stimulates adrenocorticotropin (ACTH) release and increases proopiomelanocortin mRNA levels in anterior pituitary cells by enhancing cyclic AMP (cAMP)‐dependent protein kinase activity. The phorbol ester phorbol 12‐myristate 13‐acetate (PMA) evokes these same responses from anterior pituitary cells by activating protein kinase C. Both protein kinases most likely induce their cellular effects by catalyzing the phosphorylation of specific proteins. To elucidate the mechanisms by which cAMP‐dependent protein kinase and protein kinase C promote ACTH secretion and synthesis, the phosphoproteins regulated by forskolin and PMA were identified in the cell line AtT‐20, which consists of a homogeneous population of corticotrophs. Phosphoproteins were analyzed in different subcellular fractions by two‐dimensional polyacrylamide gel electrophoresis and autoradiography. Forskolin increased phosphate incorporation into two proteins in the cytoplasmic fraction of 24 kilodaltons (kd) (pi 6.8) and 40 kd (pi 5.8), two proteins in the plasma membrane fraction of 32 kd (pi 8.3) and 60 kd (pi 8), and one protein in the nuclear fraction of 20 kd (pI 8.7). Insertion of the inhibitor of cAMP‐dependent protein kinase into the AtT‐20 cells, using a liposome technique, blocked the rise in phosphate incorporation induced by forskolin. PMA also stimulated phosphate incorporation into proteins in AtT‐20 cells. PMA increased the phosphorylation of three cytoplasmic proteins of 25 kd (pi 7.6), 40 kd (pi 5.8), and 40 kd (pI 8.1) as well as two membrane proteins of 32 kd (pi 8.3) and 60 kd (pi 8) and one nuclear protein of 20 kd (pi 6.3). The inhibitor of cAMP‐dependent protein kinase did not reduce the protein phosphorylation induced by PMA. The possible role of protein phosphorylation in the stimulation of ACTH release and synthesis by cAMP‐dependent protein kinase and protein kinase C is discussed.