bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of Opioid Receptor Subtypes in Solution
Authors: Demoliou‐Mason Catherine D. , Barnard Eric A.
Published: October 2006

Abstract: Stable opioid receptor binding activity that retains distinct subtype specificities (μ, 6, and K) has been obtained in high yields in digitonin extracts of rat brain membranes that had been preincubated with Mg2+ prior to solubilization. The dependence on Mg2+ ions for receptor activity is also expressed in the soluble state, where the presence of Mg2+ leads to high‐affinity and high‐capacity opioid peptide binding to the δ, μ, and K sites (the latter subtype measured by the binding of [3H]dynorphin1–8). Binding of opiate alkaloids to soluble receptor sites is less dependent on Mg2+ than is opioid peptide binding. Soluble opioid binding activity shows the same sensitivity to Na+ ions and guanine nucleotides as the membrane‐bound receptor. The ligand‐receptor interactions give evidence of strong positive cooperativity, which is interpreted in terms of association‐dissociation of receptor subunits on ligand binding in solution. Binding of enkephalin peptides is associated with the large macromolecules present (apparent Stokes radii > 60 Å), whereas both those and several small species present (< 60 Å) bind opiate alkaloids and dynorphin1–8.
Effect of Insulin‐Induced Hypoglycemia on the Concentrations of Glutamate and Related Amino Acids and Energy Metabolites in the Intact and Decorticated Rat Neostriatum
Authors: Engelsen Bernt , Westerberg Eva , Fonnum Frode , Wieloch Tadeusz
Published: October 2006

Abstract The glutamate (Glu) terminals in rat neostriatum were removed by a unilateral frontal decortication. One to two weeks later the effects of insulin‐induced hypoglycemia on the steady‐state levels of amino acids [Glu, glutamine (Gin), aspartate (Asp), γ‐aminobutyric acid (GABA), tau‐rine] and energy metabolites (glucose, glycogen, α‐ketoglu‐tarate, pyruvate, lactate, ATP, ADP, AMP, phosphocre‐atine) were examined in the intact and decorticated neostriatum from brains frozen in situ. The changes in the metabolite levels were examined during normoglycemia, hypoglycemia with burst‐suppression (BS) EEG, after 5 and 30 min of hypoglycemic coma with isoelectric EEG, and 1 h of recovery following 30 min of isoelectric EEG. In normoglycemia Glu decreased and Gin and glycogen increased significantly on the decorticated side. During the BS period no significant differences in the measured compounds were noted between the two sides. After 5 min of isoelectric EEG Glu, Gin, GABA, and ATP levels were significantly lower and Asp higher on the intact than on the decorticated side. No differences between the two sides were found after 30 min of isoelectric EEG. After 1 h of recovery from 30 min of isoelectric EEG Glu, Gin, and glycogen had not reached their control levels. Glu was significantly lower, and Gin and glycogen higher on the decorticated side. The Asp and GABA levels were not significantly different from control levels. The results indicate that the turnover of Glu is higher in the intact than in decorticated neostriatum during profound hypoglycemia.
Further Characterization of Dopamine Release by Permeabilized PC 12 Cells
Authors: Ahnert‐Hilger Gudrun , Gratzl Manfred
Published: October 2006

Abstract: Rat pheochromocytoma cells (PC 12) permeabilized with staphylococcal α‐toxin release [3H]dopamine after addition of micromolar Ca2+. This does not require additional Mg2+‐ATP (in contrast to bovine adrenal medullary chromaffin cells). We also observed Ca2+‐dependent [3H]‐dopamine release from digitonin‐permeabilized PC 12 cells. Permeabilization with α‐toxin or digitonin and stimulation of the cells were done consecutively to wash out endogenous Mg2+‐ATP. During permeabilization, ATP was removed effectively from the cytoplasm by both agents but the cells released [3H]dopamine in response to micromolar Ca2+ alone. Replacement by chloride of glutamate, which could sustain mitochondrial ATP production in permeabilized cells, does not significantly alter catecholamine release induced by Ca2+. However, Mg2+ without ATP augments the Ca2+‐induced release. The release was unaltered by thiol‐, hydroxyl‐, or calmodulin‐interfering substances. Thus Mg2+‐ATP, calmodulin, or proteins containing ‐SH or ‐OH groups are not necessary for exocytosis in permeabilized PC 12 cells.
Primary Structure and Tissue Distribution of Guinea Pig Gastrin‐Releasing Peptide
Authors: Shaw C. , Thim L. , Conlon J. M.
Published: October 2006

Abstract: The primary structure of gastrin‐releasing peptide from the guinea pig stomach has been determined by automated Edman degradation and shown to be identical to porcine gastrin‐releasing peptide. Extracts of guinea pig brain and small intestine contained both gastrin‐releasing peptide and its COOH‐terminal decapeptide (neuromedin C) but the stomach extracts contained only gastrin‐releasing peptide. Within the small intestine, highest concentrations of gastrin‐releasing peptide‐like immunoreactivity were found in extracts of the circular and longitudinal smooth muscle layers.
Ganglioside GM1 Causes Expression of Type B Monoamine Oxidase in a Rat Clonal Pheochromocytoma Cell Line, PC12h
Authors: Naoi Makoto , Suzuki Hiroko , Takahashi Tsutomu , Shibahara Keiichi , Nagatsu Toshiharu
Published: October 2006

Abstract: The effects of ganglioside supplementation of culture medium on monoamine oxidase (MAO) type A and B activities in a rat clonal pheochromocytoma cell line, PC12h, were examined. The MAO activity in PC12h cells proved to be mainly due to type A MAO, and type B MAO activity was negligible. After supplementation of the culture medium with ganglioside GM1, the PC 12 cells were found to express type B MAO activity after 4 days of culture, and the amount of type B activity increased with the number of days of culture. After 3 weeks of culture in the presence of GM1, type B activity was about 10% of the total, whereas in control cells type B MAO activity was only about 0.6% of the total. By kinetic analyses of type A and B MAO in PC12h cells after 3 weeks of culture, the increase of type B MAO activity was found to be due to the increase in amount of type B MAO; the Km values were almost the same and only the Vmax values were increased in the cells supplemented with GM1. Among gangliosides tested GM1 was the most effective in causing expression of type B MAO activity, whereas nerve growth factor was not effective. These results suggest that GM1 and other gangliosides may be involved in the expression of type B MAO in nerve cells and in the regulation of levels of the biogenic amines in the brain.
Intracellular pH and Catecholamine Synthesis in Cultured Bovine Adrenal Medullary Cells: Effect of Extracellular Na+ Removal
Authors: Yanagihara Nobuyuki , Yokota Kenji , Wada Akihiko , Izumi Futoshi
Published: October 2006

Incubation of cultured bovine adrenal medullary cells in Na+‐free sucrose medium or in Na+‐free Cs+ medium enhanced the synthesis of 14C‐catecholamines from [14C]tyrosine about two‐ to threefold or sixfold, respectively. The increment of 14C‐catecholamine synthesis produced by Na+‐free medium was partially dependent on the presence of Ca2+ in the medium. Dibutyrylcyclic AMP also stimulated the synthesis of 14C‐catecholamines in adrenal medullary cells, and the effects of Na+ removal and dibutyryl cyclic AMP (5 mM) on the synthesis were almost additive. The intracellular pH measured by using a weak acid 5, 5‐dimethyloxazolidine‐2, 4‐dione was 7.14 in control cells and when Na+ was replaced by sucrose or Cs+, it shifted down to 6.56 or 5.66, respectively. The fall in intracellular pH and the stimulation of 14C‐catecholamine synthesis were similarly dependent on the concentration of Na+ in the medium. The optimal pH of soluble tyrosine hydroxylase was 5.5–6.0 both in control cells and in cells incubated in Na+‐free med um. These results suggest that removal of extracellular Na+ increases the synthesis of catecholamines, at least in part, by shifting the intracellular pH toward the optimal pH of tyrosine hydroxylase.
Increased Concentrations of 3‐Hydroxykynurenine in Vitamin B6 Deficient Neonatal Rat Brain
Authors: Guilarte Tomas R. , Wagner Henry N.
Published: October 2006

Increased concentrations of the endogenous tryptophan metabolite 3‐hydroxykynurenine (3‐HK) were measured in the brains of vitamin B6 deficient neonatal rats. Mean concentrations of 3‐HK in B6 deficient cerebellum, corpus striatum, frontal cortex, and pons/medulla ranged from 9.7 to 18.6 and 102 to 142 nmol/g of wet tissue at 14 and 18 days of age, respectively. 3‐HK was not significantly increased in control neonatal or adult rat brain, vitamin B6 deficient rat brain at 7 days of age, or in brains from adult rats deprived of vitamin B6 for 58 days. The administration of daily intraperitoneal injections of vitamin B6 from the 14th to the 18th day of age decreased the concentration of 3‐HK to control levels. 3‐HK has been shown by other investigators to produce seizures when injected into the cerebral ventricles of adult rodents. Thus, our studies show the accumulation in brain of a putative endogenous convulsant as the result of a nutritional deficiency.
Depletion of myo‐Inositol and Amino Acids in Galactosemic Neuropathy
Authors: Nishimura Chihiro , Lou Marjorie F. , Kinoshita Jin H.
Published: October 2006

Abstract: : A depletion of not only myo‐inositol (MI) but also taurine and other amino acids was observed in the sciatic nerve of a galactosemic rat. Treatment of the galactosemic rats with sorbinil, an aldose reductase (AR) inhibitor, was found to block galactitol formation and protect against the loss of MI, taurine, and other amino acids. Incubation studies of sciatic nerve have revealed that [3H]MI and [3H]‐taurine were actively taken up and concentrated. Incubation of the nerve in a high‐galactose medium showed a decrease in the accumulation of [3H]MI and [3H]taurine whereas the galactitol level increased. Time‐course studies have shown that the galactitol level reached a plateau before a substantial decrease in the accumulation of [3H]MI and [3H]taurine occurred. The addition of AR inhibitors in the galactose medium significantly protected against the loss in the capacity of the nerve to accumulate [3H]MI and [3H]taurine. Hypertonicity of the galactose medium also seemed to have a protective effect similar to that of AR inhibitors.
ATP Release from Affinity‐Purified Rat Cholinergic Nerve Terminals
Authors: Richardson Peter J. , Brown Susan J.
Published: October 2006

Abstract: Cholinergic nerve terminals were affinity purified from rat caudate nucleus. On stimulation with both 22.6 mM KCl and 50 μM veratridine, ATP was released in a Ca2+‐dependent manner. The molar ratio of released acetyl‐choline to ATP (9:1) was closer to that found in isolated cholinergic vesicles (7:1) than whole terminals (3:1). Extracellular [14C]ATP was rapidly metabolized by these terminals to adenosine and inosine via ectonucleotidases. The terminals had a saturable, high‐affinity uptake mechanism for adenosine (Km= 16.6 μM). Veratridine stimulation also caused the Ca2+‐dependent release of nucleosides in a dipyridamole‐sensitive manner. Both theophylline treatment and inhibition of extracellular ATP breakdown resulted in increased ATP and nucleoside release. Extracellular adenosine was shown to inhibit acetylcholine release, probably via the Ai receptor. The role of extracellular purines at the cholinergic nerve terminal is discussed.
Regional Cerebral Glucose Utilization During Hyperglycemia
Authors: Duckrow Robert B. , Bryan Robert M.
Published: October 2006

Abstract: Previous data indicate that regional cerebral blood flow (rCBF) decreases during acute and chronic hyperglycemia. To test the hypothesis that the decrease in rCBF is secondary to a decrease in cerebral metabolic rate, the rate of regional cerebral glucose utilization (rCMRgl) was measured in awake‐restrained rats during acute and chronic hyperglycemia. Acute hyperglycemia was produced by intraperitoneal injection of glucose, and chronic hyperglycemia was produced by treatment with streptozotocin. The rCMRgl was measured over a 10‐min period using [6‐14CJglucose. Glucose utilization was normal during acute hyperglycemia but decreased by 13% following 3 weeks of chronic hyperglycemia. The absence of a decrease in rCMRgl measured during acute hyperglycemia indicates that decreased rCBF cannot be explained by a change in the metabolic rate of the brain. The decrease in rCMRgl measured during chronic hyperglycemia does not necessarily indicate the presence of a drop in the metabolic rate of the brain because ketone bodies are available as an alternate fuel for oxidative metabolism. Therefore, it is unlikely that the decrease in rCMRgl measured during chronic hyperglycemia accounts for decreased rCBF.
Ammonia‐Induced Release of Neurotransmitters from Rat Brain Synaptosomes: Differences Between the Effects on Amines and Amino Acids
Authors: Erecinska Maria , Pastuszko Anna , Wilson David F. , Nelson David
Published: October 2006

The effect of NH4Cl on release of amine and amino acid transmitters from rat brain synaptosomes was investigated. Ammonia (0.1–10 mM) stimulated the secretion of dopamine and 5‐hydroxytryptamine in a dose‐dependent manner, in a process which was additive with the effect of 40 mM K+, almost unaffected by withdrawal of Ca2+, and markedly decreased by increasing [H+] in the medium. The NH4Cl‐induced dopamine efflux, in contrast to that caused by high [K+]e, was inhibited by benztropine. The release of γ‐aminobutyric acid, aspartate, and glutamate was unaltered by [NH4Cl] < 5 mM, but somewhat stimulated at higher levels. Transmembrane pH gradient, acid inside, was dissipated by NH4Cl in a concentration‐dependent manner and the internal alkalinization correlated with the stimulation of the rate of dopamine efflux. Transmembrane electrical potential was unaffected by [ammonia] < 5 mM, but a small depolarization was observed at higher levels. It is postulated that ammonia‐induced alkalinization of the intrasynaptic storage granules causes extrusion of amines into the cytoplasm and their subsequent leakage into the medium through a reversal of the plasma membrane transporters. A lack of correlation between the release of amino acid neurotransmitters and the dissipation of the ΔpH suggests that in rat brain intrasynaptic vesicles, acidic inside, are unlikely to store substantial amounts of γ‐aminobutyric acid, aspartate, or glutamate.
Synthesis and High Affinity Uptake of Serotonin and Dopamine by Human Y79 Retinoblastoma Cells
Authors: Yorek Mark A. , Strom David K. , Spector Arthur A.
Published: October 2006

Abstract: Human Y79 retinoblastoma cells are capable of synthesizing the putative retinal neurotransmitters dopamine and serotonin. Separation of the catecholamines and indolamines by high performance liquid chromatography combined with electrochemical detection showed that the cells readily convert tyrosine to 3,4‐dihydroxyphenylalanine (DOPA) and, to a lesser extent, dopamine. When DOPA was added, a large quantity of dopamine was produced, as well as norepinephrine, epinephrine, and 3,4‐dihydroxyphenylacetic acid. Exogenous tryptophan added to the cells was partially converted to 5‐hydroxytryptophan and serotonin. A larger quantity of serotonin was produced when 5‐hydroxytryptophan was added. Y79 cells have a high and low‐affinity uptake system for dopamine and serotonin. The K′m and V′max for the high‐affinity uptake of dopamine and serotonin are 2.34 ± 0.64 and 3.63 ± 1.15 μM and 4.77 ± 1.12 and 3.20 ± 1.20 pmol min‐1 mg protein‐1, respectively. These kinetic parameters are similar to those reported for other retinal preparations where dopamine and serotonin have been suggested to function as neurotransmitters. Tyrosine and tryptophan, the physiologic precursors of dopamine and serotonin, respectively, and phenylalanine are also taken up by high and low‐affinity transport systems. The kinetic parameters for their high‐affinity uptake systems are all very similar, suggesting that they may be taken up by the same transporter. These studies show that a tumor cell line derived from the human retina synthesizes dopamine and serotonin and has high affinity uptake systems for these compounds and their pre cursors. This is consistent with the possibility that dopamine and serotonin may function as neurotransmitters in the human retina and that the Y79 retinoblastoma may be a suitable cellular model system in which to study their transport and metabolic properties.
Endopeptidase‐24.15 Is the Primary Enzyme that Degrades Luteinizing Hormone Releasing Hormone Both In Vitro and In Vivo
Authors: Molineaux Christopher J. , Lasdun Abraham , Michaud Charlene , Orlowski Marian
Published: October 2006

Abstract: The concentration of luteinizing hormone releasing hormone (LHRH) (pGlu‐His‐Trp‐Ser‐Tyr‐Gly‐Leu‐Arg‐Pro‐Gly‐NH2), which reaches the anterior pituitary via the hypothalamo‐hypophyseal portal system, appears to be controlled in part by the rate of LHRH degradation within the hypothalamus and/or pituitary. Specific, active site‐directed endopeptidase inhibitors synthesized in our laboratory were used to identify the enzyme(s) involved in LHRH degradation by hypothalamic and pituitary membrane preparations, and by an intact anterior pituitary tumor cell line (AtT20). Incubation of LHRH with pituitary and hypothalamic membrane preparations led to the formation of pGlu‐His‐Trp (LHRH1–3) as the main reaction product. Under the same conditions, addition to the incubation mixtures of captopril, an inhibitor of the angiotensin converting enzyme, led to accumulation of pGlu‐His‐Trp‐Ser‐Tyr (LHRH1–5) and, to a lesser extent, pGlu‐His‐Trp‐Ser‐Tyr‐Gly (LHRH1–6). The degradation of LHRH and the formation of the N‐terminal tri‐ and pentapeptides was blocked by N‐[1‐(R,S)‐carboxy‐3‐phenylpropyl]‐Ala‐AlaPhe‐p‐aminobenzoate (cFP‐AAF‐pAB), a specific, active site directed inhibitor of endopeptidase‐24.15. Some inhibition of LHRH degradation and formation of the N‐terminal hexapeptide was also obtained in the presence of N‐[1‐carboxy‐2‐phenylethyl]‐Phe‐p‐aminobenzoate (cFE‐F‐pAB), an inhibitor of endopeptidase‐24.11. Similar results were obtained with AtT20 cell membranes and with intact AtT20 cells in monolayer culture. Following cleavage by endopeptidases the C‐terminal part of LHRH was rapidly degraded by aminopeptidases. Superactive analogs of LHRH in which Gly6 was replaced by a D‐amino acid are resistant to degradation by both endopeptidase‐24.11 and ‐24.15. In vivo, when LHRH was injected directly into the third ventricle of rats, the presence of cFP‐AAF‐pAB inhibited LHRH degradation. It is concluded that LHRH degradation is primarily initiated by the membrane‐bound form of endopeptidase‐24.15 to yield pGlu‐His‐Trp‐Ser‐Tyr and to a lesser extent by endopeptidase‐24.11 to yield pGlu‐His‐Trp‐Ser‐Tyr‐Gly.
γ‐Aminobutyric Acid Function in the Rat Striatum Is Under the Double Influence of Nigrostriatal Dopaminergic and Thalamostriatal Inputs: Two Modes of Regulation?
Authors: Samuel Denise , Kumar Ujendra , Nieoullon André
Published: October 2006

Abstract: Glutamic acid decarboxylase (GAD), γ‐[3H]‐aminobutyric acid ([3H]GABA) high‐affinity uptake into synaptosomes, and endogenous GABA content were measured in the rat striatum 2–3 weeks following 6‐hydroxydopamine injection in the ipsilateral substantia nigra to destroy the nigrostriatal dopaminergic pathway and after kainic acid injection into the centromedial‐parafascicular complex of the ipsilateral thalamus to lesion the thalamostriatal input. Both lesions resulted in apparent GAD increase concomitant with a decreased [3H]GABA uptake into striatal synaptosomes. GABA content was increased selectively following the dopaminergic lesion. Kinetic analysis of the uptake process for [3H]GABA showed selectively a decreased Vmax following the dopaminergic lesion; in animals with thalamic lesion, however, the change only concerned the Km, which showed a decreased affinity of the transport sites for [3H]GABA. Determination of Km and Vmax for GAD action on its substrate glutamic acid showed an increased affinity of GAD for glutamic acid in the case of the dopaminergic lesion without any change in Vmax, whereas the thalamic lesion resulted in GAD increase concomitant with a selective increase in Vmax. These data suggest that striatal GABA neurons are under the influence of nigrostriatal dopaminergic neurons which may reduce the GABA turnover, whereas the exact nature of the powerful control also revealed on these neurons following thalamic lesion remains to be determined. Both lesions induced adaptive neurochemical responses of striatal GABA neurons, possibly reflecting in the case of the dopaminergic deprivation an increased GABA turnover.
Restoration of Catecholamine Content of Previously Depleted Adrenal Medulla In Vitro: Importance of Synthesis in Maintaining the Catecholamine Stores
Authors: Wakade Arun R. , Wakade Taruna D. , Malhotra Ravindra K.
Published: October 2006

Abstract: The functional integrity of adrenal chromaffin storage vesicles was studied in the perfused rat adrenal gland subjected to intense exocytosis. Continuous perfusion with 55 mM K+‐Krebs solution produced a large and uninterrupted secretion of catecholamines. Total amounts secreted within 45 min were 4.66 μg and represented almost 30% of the total tissue catecholamine content. If perfusion with excess K+ was extended to 90 min, the secretion increased further to 5.76 μg. Despite such a large secretory response, the catecholamine content of the K+‐stimulated adrenal medulla was comparable to that of unstimulated control, suggesting an enhanced resynthesis to maintain the normal levels. Pretreatment of rats with α‐methyl‐p‐tyrosine, and including this agent in the perfusion medium during stimulation with K+, caused a marked reduction in catecholamine content. The degree of depletion depended on the extent of stimulation with K+ (45% in 45 min and 60% in 90 min). Although depleted catecholamine stores did not show spontaneous recovery in 2 h, inclusion of tyrosine, l‐3,4‐dihydroxyphenylalanine or dopamine (but not epinephrine or norepinephrine) completely restored the catecholamine content of previously depleted adrenal me dulla. Repletion achieved by tyrosine was time dependent (evident in 30 min and maximum in 2 h) and blocked by α‐methyl‐p‐tyrosine but not by calcium deprivation. The ratio of epinephrine to norepinephrine remained constant during various stages of the experiment, suggesting both types of vesicles were equally affected by different treatments. The secretory response (10 Hz for 30 s) was unaffected even though tissue catecholamine stores were significantly depleted (50%). In summary, we have demonstrated that catecholamine content of the isolated perfused adrenal gland can be reduced by stimulation of exocytotic secretion in the presence of tyrosine hydroxylase inhibitor. Since the depleted stores can be fully refilled by synthesis of catecholamines from its precursors, it is suggested that chromaffin vesicles may be reutilized for the purpose of synthesis, storage, and secretion of adrenal medullary hormones.
Metalloendoprotease Cleavage of 18.2‐ and 14.1‐Kilodalton Basic Proteins Dissociating from Rodent Myelin Membranes Generates 10.0‐ and 5.9‐Kilodalton G‐Terminal Fragments
Authors: Chantry Andrew , Earl Christopher , Groome Nigel , Glynn Paul
Published: October 2006

Rat and guinea pig myelin membranes were incubated at physiological ionic strength with millimolar concentrations of Ca2+/Mg2+ ions (37°C; pH 7.4). After 1‐3 h, electrophoresis of the membranes revealed loss of 50% of 18.2‐ and 14.1‐kilodalton (kDa) forms of myelin basic protein (MBP). Concomitantly, peptides representing 25% of the original membrane‐associated MBP were detected in incubation media. Roughly equal amounts of MBP fragments with molecular masses of 10.0 and 8.4 kDa were found in media from guinea pig myelin incubations. Media from rat myelin experiments contained a major 8.4‐kDa and minor 10.0‐ and 5.9‐kDa MBP peptides. Kinetic studies implied that proteolysis occurred subsequent to MBP dissociation from the membranes. Immunoblotting studies indicated that both the 18.2‐and 14.1‐kDa forms of MBP were cleaved near residue 73 to produce a 10.0‐ and 5.9‐kDa C‐terminal fragment, respectively. Degradation of MBP in myelin membranes was partially inhibited by only 5‐20% using leupeptin (20 μM), N‐ethylmaleimide (10 mM), phenylmethylsulphonyl fluoride (1 mM), and phos‐phoramidon (50 μM) but up to 50% by dithiothreitol (DTT, 10 mM). Only DTT and 1,10‐phenanthroline substantially blocked the formation of the characteristic 10.0‐and 5.9‐kDa C‐terminal fragments. This suggests that MBP, dissociating from myelin membrane preparations, is cleaved near residue 73 by a metalloendoprotease distinct from N‐ethylmaleimide/leupeptin‐sensitive calpains and phosphoramidon‐sensitive endopeptidase 24.11.
Cyclic Nucleotides and the Activity of Glia Maturation Factor in the Hypoplastic Cerebellum of Developing Jaundiced Gunn Rats
Authors: Aono Sachiko , Kato Taiji , Tanaka Ryo , Sato Hiroshi , Semba Reiji , Kashiwamata Shigeo
Published: October 2006

Developmental changes of cyclic nucleotides were studied in the hypoplastic cerebellum of jaundiced Gunn rats over the period of postnatal days 8 to 30. The mitogenic activity of glia maturation factor was also measured at day 15. In jaundiced homozygotes(jj), the amount of cyclic GMP on a protein basis was not significantly different from that in control heterozygotes (j+) at either day 8 or 15, but at day 30 it was reduced to about 19% of the control. On the other hand, a lowered nucleotide level on a wet weight basis in jj rats was already statistically significant at day 15. In contrast to cyclic GMP, the rates of increase of cyclic AMP on a wet weight basis were almost the same in the two groups of rats, but the nucleotide levels on a protein basis at days 15 and 30 were a little, but significantly, higher in jj rats than in j+ rats. The activity of glia maturation factor in jj rats was found to be 1.5‐3 times as high as that in j+ rats. Possible implications of the present results are discussed.
In Vivo Voltammetric Study of 6‐Hydroxydopamine‐Induced Neuronal Degradation
Authors: Nakazato Taizo , Akiyama Akitane
Published: October 2006

Abstract: Freely moving rats were injected intraventricu‐larly with 200 μg of 6‐hydroxydopamine (6‐OHDA) and examined for transport of this substance to the striatum, degradation of dopamine (DA) neurons, and functional recovery through microcomputer‐controlled in vivo voltam‐metry. Approximately 6 min after the injection, 6‐OHDA began to appear in the extracellular fluid of the contralat‐eral striatum. It increased linearly and began to decrease exponentially with the termination of the injection. Two hours after the injection with 6‐OHDA, a peak began to appear at the same potential as the peak of DA in the differential pulse voltammogram. It persisted for approximately 3 days. When the rats were injected intraperitoneally with L‐3,4‐dihydroxyphenylalanine (l‐Dopa), the conversion of l‐Dopa to DA was not found 1 week after the 6‐OHDA injection, but was clearly recognized 5 months after the injection. When the rats were examined for behavioral changes arising from the l‐Dopa injection, they were found to be clearly less hyperactive 5 months after the 6‐OHDA injection than 1 week after.
Glial Fibrillary Acidic Protein Increases in the Spinal Cord of Lewis Rats with Acute Experimental Autoimmune Encephalomyelitis
Authors: Aquino Dennis A. , Chiu Fung‐Chow , Brosnan Celia F. , Norton William T.
Published: October 2006

Abstract: Glial fibrillary acidic protein (GFAP) in the spinal cords of Lewis rats with acute experimental autoimmune encephalomyelitis (EAE) was quantitated by densi‐tometry of both stained gels and immunoblots of electro‐phoretically separated cytoskeletal proteins. The experimental period ranged from 7 to 65 days postinocula‐tion (dpi). Greater than 92% of the total spinal cord GFAP was recovered in the Triton‐insoluble cytoskeletal pellet; less than 2% was truly soluble. GFAP increased gradually and significantly with time, reaching a level one‐and‐a‐half to two times greater than that of controls by 35 dpi and remaining elevated at 65 dpi. In EAE animals, GFAP was 33% of the total Triton‐insoluble protein (excluding his‐tones and other small basic proteins) at 7 dpi, rising to 48% at 65 dpi. Increases in vimentin were also noted, following a time course similar to that of GFAP. An increase in immu‐nocytochemical staining of GFAP was noticeable at 10 dpi and became marked at 14 dpi, a time before GFAP levels had increased significantly. Thus, enhanced staining at the peak of the disease cannot be explained simply by an increase in antigen protein. Other possible explanations, such as an increase in soluble GFAP content, proteolytic degradation, or modifications in the immunochemical properties of GFAP in EAE animals, were ruled out. Both the biochemical and immunocytochemical increases in GFAP persisted through 65 dpi, even though the animals recovered from clinical signs at approximately 18 dpi.
Activation of the Arachidonate 5‐Lipoxygenase Pathway in the Canine Basilar Artery After Experimental Subarachnoidal Hemorrhage
Authors: Shimizu Takao , Watanabe Takashi , Asano Takao , Seyama Yousuke , Takakura Kintomo
Published: October 2006

Abstract: Severe cerebral vasospasm as confirmed by an‐giography was induced in dogs by injection of autologous blood into the cisterna magna, and the resultant leukotriene formation in the isolated basilar artery was examined. When stimulated with calcium ionophore (A 23187), the arteries of the treated animals produced a significant amount of leu‐kotrienes B4 (85 ± 12 pmol/mg protein, n = 3) and C4 (72 ± 14 pmol/mg), in addition to 5(S)‐hydroxy‐6,8,l 1,14‐ei‐cosatetraenoic acid. Structural elucidations of these metabolites were performed by radioimmunoassays or gas chro‐matography‐mass spectrometry, following purification with HPLC. The artery of the untreated dog produced none of these compounds from either exogenous or endogenous ar‐achidonic acid, under stimulation with the calcium ionophore. However, the homogenates from both animals converted exogenous leukotriene A4 to leukotrienes B4 and C4. These observations suggest that the normal basilar artery contains no detectable amount of 5‐lipoxygenase, and that a prominent activation of this enzyme occurred (2.1 nmol 5‐HETE/5 min/mg of protein) after subarachnoidal hemorrhage. The observation that fatty acid hydroperoxides stimulated the 5‐lipoxygenase activity indicates a possible role of lipid peroxides in the development of vasospasm.
Fast HPLC Estimation of γ‐Aminobutyric Acid in Microdialysis Perfusates: Effect of Nipecotic and 3‐Mercaptopropionic Acids
Authors: Kehr Jan , Ungerstedt Urban
Published: October 2006

Abstract: A method for rapid, automated (<5 min), and sensitive (detection limit 50 fmol/10 μl) determination of γ‐aminobutyric acid (GABA) is described. The method is based on precolumn derivatiza‐tion with o‐phthaldialdehyde/t‐butylthiol reagent and separation by reverse‐phase HPLC with electrochemical detection under isocratic conditions. A 100 × 4 mm Nucleosil 3 C18 column was used; the mobile phase consisted of 0.15 M sodium acetate, 1 mM EDTA (pH 5.4), and 50% acetonitrile; the flow rate was 0.8 ml/min. The potential of the glassy carbon working electrode was +0.75 V. The method allows for the monitoring of GABA levels in the extracellular fluid sampled by microdialysis as documented in the present study when 0.5 mM nipecotic acid is infused via the probe, or 3‐mercaptopro‐pionic acid is injected at a dose of 100 mg/kg i.p. There was a 15‐fold increase of extracellular GABA after nipecotic acid, whereas in the second case the inhibition of GABA synthesis was followed by a 74% decrease of GABA as compared to basal levels.
[3H]Dihydrotetrabenazine, a New In Vitro Monoaminergic Probe for Human Brain
Authors: Scherman Daniel , Raisman Rita , Ploska Alain , Agid Yves
Published: October 2006

Abstract: The monoamine transporter of dopamine (DA), noradrenaline, and 5‐hydroxytryptamine synaptic vesicles was assayed in rat and human brain homogenates by in vitro binding of [3H]dihydrotetrabenazine. [3H]Reserpine, a second ligand of the vesicular monoamine transporter, could not be used. [3H]Dihydrotetrabenazine binding in rat brain was stable after 72 h at 22°C postmortem. In major human brain regions, [3H]dihydrotetrabenazine binding was specific and saturable (KD, 2.7 nM). Displacement constants by substrates or inhibitors of vesicular monoamine uptake, and regional distribution in human brain were similar to those found in rodents. The highest densities of binding sites were observed in caudate nucleus, putamen, and accumbens nucleus. In caudate nucleus and in putamen from normal human subjects, [3H]dihydrotetrabenazine binding and homovanillic acid concentration were significantly or nearly significantly correlated. A weaker correlation was found between [3H]dihydrotetrabenazine binding and DA, in association with a higher variability of DA. [3H]Dihydrotetrabenazine binding in caudate nucleus and in putamen decreased significantly with age, unlike DA and homovanillic acid concentrations. The results establish [3H]dihydrotetrabenazine as a presynaptic monoaminergic ligand of interest for studies on postmortem human brain.
Evidence that Tobacco Smoking Increases the Density of (−)‐[3H]Nicotine Binding Sites in Human Brain
Authors: Benwell Maureen E. M. , Balfour D. J. K. , Anderson J. M.
Published: October 2006

Abstract: In a postmortem study of nicotinic receptors in human brain, cigarette smoking was found to be associated with increased (−)‐[3H]nicotine binding to membranes prepared from gyrus rectus (Brodmann area 11) (p < 0.001), hippocampal neocortex (Brodmann area 27), cerebellar cortex (p < 0.01), hippocampal formation (Ammon's horn + subiculum), and the median raphe nuclei of the midbrain (p < 0.05) but not the medulla oblongata. Analysis of the binding data suggested that the increased binding reflected an increase in the density of the receptors rather than a change in their affinity for (−)‐nicotine. The effects of smoking were not influenced significantly by either the sex or age of the subject. It is concluded that smoking evokes an increase in high‐affinity nicotine binding similar to that observed previously in animals treated chronically with nicotine and that the effect of smoking on these sites is probably caused by the nicotine present in the tobacco smoke.
Regional Alterations in Blood‐to‐Brain Transfer of α‐Aminoisobutyric Acid and Sucrose, After Chronic Administration and Withdrawal of Dexamethasone
Authors: Ziylan Y. Z. , Lefauconnier J. M. , Bernard G. , Bourre J. M.
Published: October 2006

Abstract: The effect of dexamethasone administration and withdrawal was studied with respect to blood‐brain barrier function. The tracers α‐[3H]aminoisobutyric acid (AIB) (MW 104) and [14C]sucrose (MW 342), which have a low permeability across the intact endothelium, were simultaneously injected intravenously in rats treated with dexamethasone and placebo‐treated control animals or in rats in which dexamethasone treatment was discontinued 3 days before the experiment. Unidirectional transfer constants (Ki) were determined in discrete brain regions. Steroid administration reduced the rate of influx of AIB and sucrose, whereas discontinuation of drug resulted in an increased permeability. These findings suggest that when exposure to glucocorticoids is prolonged, the efficiency of medical treatment of CNS diseases may decrease due to reduction of drug delivery to CNS. Thus, these experimental findings may have particular importance in the clinical setting of drug administration when considering the combination of steroids with other drugs, and may aid in understanding better the pathogenesis of some types of brain edema seen in patients from whom corticosteroid therapy has been withdrawn.
Solubilization of the N‐Methyl‐d‐Aspartate Receptor Channel Complex from Rat and Porcine Brain
Authors: McKernan Ruth M. , Castro Sandra , Poat Judith A. , Wong Erik H. F.
Published: October 2006

Abstract: The N‐methyl‐D‐aspartate (NMDA) receptor complex as defined by the binding of [3H]MK‐801 has been solubilized from membranes prepared from both rat and porcine brain using the anionic detergent deoxycholate (DOC). Of the detergents tested DOC extracted the most receptors (21% for rat, 34% for pig), and the soluble complex, stabilized by the presence of MK‐801, could be stored for up to 1 week at 4°C with <25% loss in activity. Receptor preparations from both species exhibited [3H]MK‐801 binding properties in solution very similar to those observed in membranes (Bmax= 485 ± 67 fmol/mg of protein, KD= 11.5 ± 2.9 nM in rat; Bmax= 728 ± 108 fmol/mg of protein, KD= 7.1 ± 1.6 nM in pig, n = 3). The pharmacological profile of the solubilized [3H]MK‐801 binding site was virtually identical to that observed in membranes. The rank order of potency of: MK‐801 > (‐)‐MK‐801 = thienylcyclohexylpiperidine > dexoxadrol > SKF 10,047 > ketamine, for inhibition of [3H]MK‐801 binding, was observed in all preparations. The receptor complex in solution exhibited many of the characteristic modulations observed in membranes. After removal of small‐molecular‐weight endogenous substances by gel filtration, the following effects were observed: (1) NMDA receptor agonists such as L‐glutamate enhanced binding of [3H]MK‐801 in a concentration‐dependent manner; (2) glycine stimulated [3H]MK‐801 binding in the presence of submaximal concentrations of glutamate; (3) in the absence of glutamate and glycine, the divalent cations Mg2+, Ca2+, and Mn2+ stimulated [3H]MK‐801 binding (EC50: 10–100 μM), whereas in the presence of glutamate and glycine all divalent cations tested were inhibitory with a rank order of potency of Zn2+ > Cd2+ Mn2+ > Mg2+ > Ca2+. In summary, the soluble NMDA receptor, labelled at the cation channel site with [3H]MK‐801, maintains the ability to bind NMDA agonists, glycine, and divalent cations in a manner akin to that observed in membranes.
Comparative Effects of Chronic Exposure to Ethanol and Calcium Channel Antagonists on Calcium Channel Antagonist Receptors in Cultured Neural (PC12) Cells
Authors: Marks Shelley S. , Watson Daniel L. , Carpenter Celia L. , Messing Robert O. , Greenberg David A.
Published: October 2006

Abstract: Treatment with 200 mM ethanol for 6 days increased binding of the Ca2+ channel antagonist, (+)‐[3H]PN 200‐110, to intact PC12 cells in culture. Enhancement of binding by ethanol was due to an increase in binding site number without appreciable change in binding affinity. Long‐term exposure to Ca2+ channel antagonist drugs (nifedipine, verapamil, or diltiazem), which, like ethanol, acutely inhibit Ca2+ flux, failed to alter (+)‐[3H]PN 200‐110 binding to PC12 membranes. Cotreatment of ethanol‐containing cultures with the Ca2+ channel agonist, Bay K 8644, did not attenuate the response to ethanol; instead, chronic exposure to Bay K 8644 alone increased (+)‐[3H]PN 200‐110 binding. These results suggest that chronic exposure to ethanol increases Ca2+ channel antagonist receptor density in living neural cells, but that acute inhibition of Ca2+ flux by ethanol is unlikely to trigger this response.
Effects of Microdialysis‐Perfusion with Anisoosmotic Media on Extracellular Amino Acids in the Rat Hippocampus and Skeletal Muscle
Authors: Lehmann Anders
Published: October 2006

Abstract: Changes in the levels of amino acids have been implicated as being important in osmoregulation both within and outside the CNS. The present study addressed the question of whether changes in osmolality affect the extracellular concentration of amino acids in the rat hippocampus and femoral biceps muscle (FBM). Microdialysis probes were implanted in these tissues and perfused with standard physiological saline. Amino acid concentrations in the dialysate were determined with HPLC separation of o‐phthaldialdehyde derivatives and fluorescence detection. The osmolarity of the perfusion buffer was gradually decreased by reduction of the concentration of NaCl from 122 to 61 to 0 mM. In other experiments, the osmolarity was increased by elevation of the NaCl level from 122 to 183 to 244 mM or by addition of mannitol. Glutamate, aspartate, γ‐aminobutyrate, and alanine levels in dialysate from the hippocampus increased when the concentration of NaCl was decreased by 61 mM, and they were further elevated when NaCl was omitted. Taurine and phosphoethanolamine (PEA) levels were maximally elevated at the intermediary decrease of NaCl concentration, and glutamine in particular but also methionine and leucine were suppressed by perfusion with hypoosmolar medium. The amino acid response of the FBM differed substantially from that of the hippocampus. The aspartate content increased slightly, and there was a marginal transient increase in PEA level. Perfusion with media containing high concentrations of NaCl induced diminished dialysate levels of taurine, PEA, and glutamate, whereas levels of other amino acids were either unaffected or increased. Mannitol administration via the perfusion fluid led to reduced levels of taurine, PEA, glutamate, and aspartate. In contrast to the effects of high NaCl levels, hyperosmotic mannitol did not induce increases in level of any of the amino acids detected. The results suggest that taurine and PEA are involved in osmoregulation in the mammalian brain. From a quantitative viewpoint, taurine seems to be most important. Transmitter amino acids may also be involved in the maintenance of the volume of neural cells subjected to severe disturbances in osmotic equilibrium.
Rat Brain Synaptosomal ATP:AMP‐Phosphotransferase Activity
Authors: Nagy Agnes K. , Shuster Terifence A. , Delgado‐Escueta Antonio V.
Published: October 2006

Abstract: Adenylate kinase activity (ATPlAMP‐phospho‐transferase; EC 2.7.4.3) was studied in various subcellular fractions of rat brain tissues. Because of the presence of other adenosine nucleotide‐utilizing enzymes, adenylate kinase activity was assayed in both the forward and rejverse directions by using coupled enzyme systems and by bsing a specific adenylate kinase inhibitor, P1,P5‐di(adenasine‐5′) penta‐phosphate. As expected, the highest specific adenylate kinase activity (2.89 μumol/min/mg of protein) was detected in the cytosolic brain fraction. However, substantial enzyme activity (0.68 μumol/min/mg) was also found in the iintact synaptosomal fraction isolated on Percoll/sucrose gradients. The increased specific enzyme activity of purified sytfaptosomes and the differences found between the kinetic parameters of the membrane‐bound and cytosolic enzyme forms suggest that the synaptosomal adenylate kinase activity cannot be attributed to the small amount of contaminating cytosol present in our preparations. The adenylate kinase enzyme adhered to purified synaptic plasma membranes and was not released by washings with isoosmotic sucrose medium. The facts that the adenylate kinase enzyme activity could be measured in intact synaptosomal preparations and that both its substrates and its inhibitors do not cross intact plasma membranes support the possibility that the synaptosomal adenylate kinase is an ecto‐enzyme.
Myelin Membrane Structure and Composition Correlated: A Phylogenetic Study
Authors: Kirschner Daniel A. , Inouye Hideyo , Ganser Allen L. , Mann Vicki
Published: October 2006

Abstract: We have correlated myelin membrane structure with biochemical composition in the CNS and PNS of a phylogenetic series of animals, including elasmobranchs, teleosts, amphibians, and mammals. X‐ray diffraction patterns were recorded from freshly dissected, unfixed tissue and used to determine the thicknesses of the lipid bilayer and the widths of the spaces between membranes at their cytoplasmic and extracellular appositions. The lipid and protein compositions of myelinated tissue from selected animals were determined by TLC and sodium dodecyl sulfate‐polyacrylamide gel elec‐trophoresis/immunoblotting, respectively. We found that(l) there were considerable differences in lipid (particularly gly‐colipid) composition, but no apparent phylogenetic trends; (2) the lipid composition did not seem to affect either the bilayer thickness, which was relatively constant, or the membrane separation; (3) the CNS of elasmobranch and teleost and the PNS of all four classes contained polypeptides that were recognized by antibodies against myelin Po glycoprotein; (4) antibodies against proteolipid protein (PLP) were recognized only by amphibian and mammalian CNS; (5) wide extracellular spaces (ranging from 36 to 48 Å) always correlated with the presence of Po‐immunoreactive protein; (6) the narrowest extracellular spaces (∼31 Å) were observed only in PLP‐containing myelin; (7) the cytoplasmic space in PLP‐containing myelin (∼31 Å) averaged ∼5Å less than that in Po‐containing myelin; (8) even narrower cytoplasmic spaces (∼24Å) were measured when both Po and 11–13‐kilodalton basic protein were detected; (9) proteins immu‐noreactive to antibodies against myelin P2 basic protein were present in elasmobranch and teleost CNS and/or PNS, and in mammalian PNS, but not in amphibian tissues; and (10) among mammalian PNS myelins, the major difference in structure was a variation in membrane separation at the cytoplasmic apposition. These findings demonstrate which features of myelin structure have remained constant and which have become specifically altered as myelin composition changed during evolutionary development.
Subacute Noradrenergic Agonist Infusions In Vivo Increase Na+, K+‐ATPase and Ouabain Binding in Rat Cerebral Cortex
Authors: Swann Alan C. , Steketee Jeffrey D.
Published: October 2006

Abstract: In order to investigate the specificity of noradrenergic effects on Na+, K+‐ATPase, we infused noradrenergic agonists into the cerebral ventricles of rats, with or without depletion of forebrain norepinephrine. Infusion of norepinephrine, isoproterenol, or phenylephrine increased ouabain binding in intact rats, whereas clonidine infusion decreased binding. Depletion of forebrain norepinephrine by destruction of the dorsal noradrenergic bundle reduced ouabain binding. Norepinephrine infusion reversed the effect of dorsal bundle lesion; isoproterenol and phenylephrine increased ouabain binding in lesioned rats, but did not restore the effect of the lesions. Clonidine had no effect in lesioned rats. Effects on Na+,K+‐ATPase activity were similar, but smaller. These results suggest that stimulation of both α1‐ and β‐noradrenergic receptors may be necessary for optimal Na+,K+‐ATPase, and that clonidine reduces Na+,K+‐ATPase indirectly through decreased norepinephrine release.