bluuebunny/crossref_metadata_2025 huggingface dataset:
Inositol Phospholipid Hydrolysis in Rat Cerebral Cortical Slices: I. Receptor Characterisation
Authors: Brown Elaine , Kendall David A. , Nahorski Stefan R.
Published: October 2006

Abstract: Characterisation of receptor‐mediated breakdown of inositol phospholipids in rat cortical slices has been performed using a direct assay which involves prelabelling with [3H]inositol. When slices were preincubated with [3H]inositol, lithium was found to greatly amplify the capacity of receptor agonists such as carbachol, noradrenaline, and 5‐hydroxytryptamine to increase the amount of radioactivity appearing in the inositol phosphates. Using a large variety of agonists and antagonists it could be shown that cholinergic muscarinic, α1,‐adrenoceptor, and histamine H1, receptors appear to be linked to inositol phospholipid breakdown in cortex. The large responses produced by receptor agonists allowed a clear discrimination between full and partial agonists as well as quantitative analysis of competitive antagonists for each receptor. Whereas carbachol and acetylcholine (in the presence of a cholinesterase inhibitor) were full agonists, oxotremorine and arecoline were only partial agonists. Very low concentrations of atropine shifted the carbachol dose‐response curve to the right and allowed inhibition constants for the antagonist to be easily calculated. The nicotinic antagonist, mecamylamine, was ineffective. Noradrenaline and adrenaline were full agonists at α1‐adrenoceptors, but phenylephrine and probably methoxamine were partial agonists. Prazosin, but not yohimbine, potently and competitively antagonised the noradrenaline inositol phospholipid response. Mepyramine but not cimetidine competitively antagonised the histamine response. These data provide strong confirmation for the potentiating effect of lithium on neurotransmitter inositol phospholipid breakdown and emphasise the ease with which functional responses at a number of cortical receptors can be characterised.
Intralaminar Neurochemical Distributions in Human Midtemporal Cortex: Comparison Between Alzheimer's Disease and the Normal
Authors: Perry Elaine K. , Atack John R. , Perry Robert H. , Hardy John A. , Dodd Peter R. , Edwardson James A. , Blessed Garry , Tomlinson Bernard E. , Fairbairn Andrew F.
Published: October 2006

Abstract: The intralaminar distributions of transmitter and nontransmitter enzyme activities and amino acid levels were determined in the midtemporal cortices from normal individuals and established cases of Alzheimer's disease. In the normal, choline acetyltransferase (CAT) and acetylcholinesterase (AChE) activities were relatively high in the outer cortical layers, particularly, for CAT, in the two granular layers (II and IV). Both activities were reduced in Alzheimer's disease at all, although generally most extensively in the outer and middle layers of the gray matter whereas activities were near normal in the white matter. Further, the enzyme distribution patterns of these cholinergic activities were also disrupted in Alzheimer's disease and the activity of CAT throughout the cortex was generally reduced to that found in the white matter. No such differences in distribution were found for two other enzymes, pseudocholin‐esterase and lactate dehydrogenase. Assessment of the γ‐aminobutyric acid (GABA) system in the normal revealed a much more extensive intralaminar variation in the enzyme, glutamate decarboxylase, compared with the level of GABA itself. In contrast with the cholinergic enzymes, neither the levels nor intralaminar patterns of GABA were altered in Alzheimer's disease. From an analysis of free amino acids at the different cortical levels, the cortical pattern of glutamic acid in the normal was different from that for GABA, asp artic acid, or non‐transmitter amino acids such as alanine. Neither of the putative amino acids, glutamate or aspartate, was altered in Alzheimer's disease. These findings demonstrate the relatively selective nature of micro chemical changes oc‐curing in the cortex in Alzheimer's disease and suggest that a functional abnormality in cholinergic input to the outer neocortical layers (I‐IV) with predominantly receptive and associative functions may be an important feature of the disease.
Aspartate Aminotransferase Activity in Fiber Tracts of the Rat Brain
Authors: Godfrey Donald A. , Bowers Marc , Johnson Brian A. , Ross C. David
Published: October 2006

Abstract: Activity of aspartate aminotransferase, an enzyme which catalyzes the interconversion of the excitatory transmitter candidates, glutamate and aspartate, has been measured in fiber tracts of rat, with an emphasis on sensory and motor systems of the brain. Most tracts had significantly higher activities than the cholinergic facial nerve root, consistent with the possibility that a component of aspartate aminotransferase activity might serve as a marker for neurons using glutamate and/or aspartate as neurotransmitter. Highest activity was in the auditory nerve root. On the other hand, a close correlation was found between aspartate aminotransferase and malate de‐hydrogenase activities in the fiber tracts, raising the question whether aspartate aminotransferase activity may be more closely related to energy metabolism than to transmitter metabolism.
Elevation of the Extracellular Concentrations of Glutamate and Aspartate in Rat Hippocampus During Transient Cerebral Ischemia Monitored by Intracerebral Microdialysis
Authors: Benveniste Helene , Drejer Jørgen , Schousboe Arne , Diemer Nils H.
Published: October 2006

Abstract: Rats were implanted with 0.3‐mm‐diameter dialysis tubing through the hippocampus and subsequently perfused with Ringer's solution at a flow rate of 2 (μ1/min. Samples of the perfusate representing the extracellular fluid were collected over 5‐min periods and subsequently analyzed for contents of the amino acids glutamate, as‐partate, glutamine, taurine, alanine, and serine. Samples were collected before, during, and after a 10‐min period of transient complete cerebral ischemia. The extracellular contents of glutamate and aspartate were increased, respectively, eight‐and threefold during the ischemic period; the taurine concentration also was increased 2.6‐fold. During the same period the extracellular content of glutamine was significantly decreased (to 68% of the control value), whereas the concentrations of alanine and serine did not change significantly during the ischemic period. The concentrations of γ‐aminobutyric acid (GABA) were too low to be measured reliably. It is suggested that the large increase in the content of extracellular glutamate and aspartate in the hippocampus induced by the ischemia may be one of the causal factors in the damage to certain neurons observed after ischemia.
Regional Distribution of Homocysteine in the Mammalian Brain
Authors: Broch Ole Jacob , Ueland Per Magne
Published: October 2006

The regional distribution of l‐homocysteine (Hcy) was determined in brains from mouse, rat, guinea pig, and rabbit, using a sensitive radioenzymatic assay. Large interspecies variations in the Hcy content in various parts of the brain were observed, but cerebellum contained the highest amount in all species investigated. In the rat the amount of Hcy in cerebellum (6.4 nmol/g) was about sixfold higher than in most other parts of the brain, whereas in the mouse and guinea pig the amount in cerebellum (about 1 nmol/g) was only twofold higher than in the other brain regions. There was a remarkably high level of Hcy in all regions of the rabbit brain (4–10 nmol/g); the highest concentration was found in the cerebellar white matter. In this species the amount of Hcy in all brain regions examined exceeded that in the liver.
Purification and Characterization of a Neuropeptide‐Degrading Aminopeptidase from Human Brain
Authors: McDermott J. R. , Mantle D. , Lauffart B , Kidd A. M.
Published: October 2006

Abstract: The major aminopeptidase from human post‐mortem brain (occipital cortex) was purified to homogeneity (as judged by polyacrylamide gel electrophoresis) by anion‐exchange chromatography (two steps) and gel filtration (two steps). The molecular weight of the enzyme was estimated as 105,000 from gel filtration. Maximum activity was obtained in the presence of 0.5 mM Ca2+ and 1 mM 2‐mercaptoethanol at pH 7.3. Enzyme activity was lost on freezing and thawing or on lyophilization. The enzyme was inhibited by metal‐ion chelating agents, sulphhydryl blocking agents, bestatin, and puromycin. A series of amino acyl‐7‐amido‐4‐methylcoumarins was hydrolysed by the enzyme, with the alanyl derivative being hydrolysed most rapidly (Km 170 μM). Specificity studies with a series of alanine dipeptides suggested that a hydrophobic second residue favoured hydrolysis. Several naturally occurring neuropeptides, including Leu5‐enkephalin (Km 180 μM), cholecystokinin octapeptide, and Arg8‐vasopressin, were hydrolysed by the aminopeptidase. In a series of opioid peptides, increasing chain length led to decreased susceptibility to hydrolysis. Sulphation of the Tyr1 residue of Leu5‐enkephalin and the Tyr2 residue of cholecystokinin octapeptide made the peptides more resistant to hydrolysis.
Circadian Rhythm in Pineal N‐Acetyltransferase Activity: Phase Shifting by Dark Pulses (III)
Authors: Binkley Sue , Mosher Karen , White Beatrix H.
Published: October 2006

Abstract: N‐Acetyltransferase (NAT) is an enzyme whose rhythmic activity in the pineal gland and retina is thought to be responsible for melatonin circadian rhythms. The enzyme has circadian properties–its rhythm persists in constant conditions, and it is precisely controlled by light and dark. Experiments are reported in which 4‐h light or dark pulses were imposed on chicks (Gallus domesticus) over a 24‐h period. Pineal NAT profiles were measured during and subsequent to the pulses. The phase of the NAT cycle following pulses was plotted to obtain phase‐response curves. Light pulses produced a maximum phase shift (advance of 5 h) 8 h after the expected time of lights‐out; dark pulses produced a maximum phase shift (advance of 4 h) 3 h after the expected time of lights‐out. Maximum phase delays (−2 h) occurred 1–2 h after the expected lights‐out for light pulses and 8 h after expected lights‐on for dark pulses.
Characterization of Functional Receptors for Vasoactive Intestinal Peptide in Bovine Cerebral Arteries
Authors: Suzuki Y. , McMaster D. , Huang M. , Lederis K. , Rorstad O. P.
Published: October 2006

Abstract: This study reports the characterization of receptors for vasoactive intestinal peptide (VIP) on membranes prepared from bovine cerebral arteries. By use of HPLC we prepared two purified monoiodinated VIP radioligands with nearly equivalent cerebral vasorelaxant potency as native VIP, [Tyr(125I)10] VIP and [Tyr(125I)22] VIP. The former resulted in a higher proportion of specific binding to arterial membranes than the latter and was therefore thought to be the superior radioligand for receptor characterization. The binding of [Tyr(125I)10]VIP to cerebral arterial membranes was saturable, specific, reversible, and dependent on time and temperature. Scatchard analysis suggested the presence of a high‐ and a low‐affinity binding site with KD values of 0.2 and 11 nM and receptor concentrations of 79 and 737 fmol/mg of protein, respectively. The dose‐response curves for binding to the VIP receptor by the VIP‐homologous peptides PHI, PHM, and rat growth hormone‐releasing factor (GRF) were very similar to their dose‐response curves for relaxation of cerebral arteries. The order of potency was VIP > PHM > PHI > rat GRF. It is suggested that the characteristics of the vascular VIP binding sites and the close correlation between the binding and vasorelaxant properties of VIP and its related peptides argue for the vascular binding sites being functional receptors for VIP.
Specific Replacements of Pyruvate for Trophic Support of Central and Peripheral Nervous System Neurons
Authors: Facci Laura , Skaper Stephen D. , Varon Silvio
Published: October 2006

Abstract: When embryonic central nervous system neurons are seeded at low densities with Eagle's basal medium supplemented with the serum substitute N1, glucose, and glutamine, neuronal survival for even 24 h requires the additional supply of exogenous pyruvate–and so does the survival of many peripheral nervous system neurons. Pyruvate can be replaced by α‐ketoglutarate or oxaloacetate, but not by Krebs cycle substrates that are not keto acids. Most other α‐keto acids tested (though not β‐ or γ‐keto acids) also mimic pyruvate. The apparent equivalence to pyruvate of all these compounds includes identical ED50 values (300 μM for embryonic avian forebrain neurons, 30–40 μM for rat hippocampal neurons), and also identical susceptibilities to the pyruvate‐sparing effects of other low‐molecular‐weight agents present in Dulbecco's modified Eagle's medium or in astroglia conditioned medium. The substitute α‐keto acids, however—unlike pyruvate, α‐ketoglutarate, or oxaloacetate—support cell survival only in the presence of α‐amino acids that transaminate to α‐ketoglutarate, oxaloacetate, or pyruvate. The α‐keto acids, therefore, operate as acceptors of amino groups from appropriate donors to generate Krebs cycle‐relevant substrates. Consistent with this view, [14C]glutamate did not generate appreciable 14CO2 unless accompanied by a suitable α‐keto acid. Thus: (1) the true survival‐controlling agents for these cultured CNS neurons are pyruvate, α‐ketoglutarate, and oxaloacetate; (2) other α‐keto acids provide for neuronal support only insofar as they make it possible for exogenously supplied α‐amino acid precursors to generate intracellularly one of the three critical agents; and (3) the inability of CNS neurons to utilize glucose for their survival resides with some restriction of substrate access to the Krebs cycle.
Decrease of Clonidine Binding Affinity to α2‐Adrenoceptor by ADP‐Ribosylation of 41,000‐Dalton Proteins in Rat Cerebral Cortical Membranes by Islet‐Activating Protein
Authors: Nomura Yasuyuki , Kitamura Yoshihisa , Segawa Tomio
Published: October 2006

Abstract: The IC50 value for inhibition of specific [3H]yohimbine binding to rat cerebral cortical membranes by clonidine was increased, and the Hill coefficient (nH) approached unity in the presence of 150 μM GTP. Pretreatment of membranes with islet‐activating protein (IAP) in the presence of NAD caused an increase in IC50 and nH values for clonidine compared with control membranes in the absence of GTP, the addition of which was without effect. Scatchard analysis showed that the Bmax value of the high‐affinity component in [3H]clonidine binding was decreased by pretreatment with IAP/NAD. GTP in a concentration range of 0.1 μM‐ I mM caused significant elevation of [3H]yohimbine binding. In IAP/NAD‐pretreated membranes, however, [3H]yohimbine binding was no longer affected by GTP, although IAP/NAD significantly (p < 0.01) increased [3H]yohimbine binding compared to control. IAP ADP‐ribosylated 41,000 dalton proteins of cerebral cortical membranes. From these results, it can be suggested that inhibitory guanine nucleotide regulatory protein with MI 41,000 couples to α2‐adrenoceptors to regulate binding affinity of agonists and antagonists in membranes of the rat cerebral cortex.
Inhibition of a Low Km GTPase Activity in Rat Striatum by Calmodulin
Authors: Treisman Glenn J. , Muirhead Nancy , Iwaniec Lynn , Gnegy Margaret E.
Published: October 2006

Abstract: In rat striatum, the activation of adenylate cyclase by the endogenous Ca2+‐binding protein, calmodulin, is additive with that of GTP but is not additive with that of the nonhydrolyzable GTP analog, guanosine‐5′‐(β, γ‐imido)triphosphate (GppNHp). One possible mechanism for this difference could be an effect of calmodulin on GTPase activity which has been demonstrated to “turn‐off” adenylate cyclase activity. We examined the effects of Ca2+ and calmodulin on GTPase activity in EGTA‐washed rat striatal particulate fractions depleted of Ca2+ and calmodulin. Calmodulin inhibited GTP hydrolysis at concentrations of 10−9–10−6M but had no effect on the hydrolysis of 10−5 and 10−6M GTP, suggesting that calmodulin inhibited a low Km GTPase activity. The inhibition of GTPase activity by calmodulin was Ca2+‐dependent and was maximal at 0.12 μM free Ca2+. Maximal inhibition by calmodulin was 40% in the presence of 10−7M GTP. The IC50 for calmodulin was 100 nM. In five tissues tested, calmodulin inhibited GTP hydrolysis only in those tissues where it could also activate adenylate cyclase. Calmodulin could affect the activation of adenylate cyclase by GTP in the presence of 3,4‐dihydroxyphenylethylamine (DA, dopamine). Calmodulin decreased by nearly 10‐fold the concentration of GTP required to provide maximal stimulation of adenylate cyclase activity by DA in the striatal membranes. The characteristics of the effect of calmodulin on GTPase activity with respect to Ca2+ and calmodulin dependence and tissue specificity parallel those of the activation of adenylate cyclase by calmodulin, suggesting that the two activities are closely related. Inhibition of a low Km GTPase activity by calmodulin could represent an action of calmodulin in increasing the association of a GTP‐binding protein with the catalytic subunit activity resulting in a reduction of the “turn‐off” GTPase activity.
Metabolic Fluxes Between [14C]2‐Deoxy‐D‐Glucose and [14C]2‐Deoxy‐D‐Glucose‐6‐Phosphate in Brain In Vivo
Authors: Huang Ming‐Ta , Veech Richard L.
Published: October 2006

Abstract: The rates of the phosphorylation and dephosphorylation of 2‐deoxyglucose were measured in rat brain in vivo using tracer kinetic techniques. The rate constant for each reaction was estimated from two separate experiments with different protocols for tracer administration. Tracer amounts of [1‐14C]2‐deoxyglucose (1 μCi) were injected through the internal carotid artery (intraarterial experiment), or through the atrium (intravenous experiment). Brains were sampled by freeze‐blowing at various times after the injection. In the intraarterial experiment, the rate constant for the forward reaction from 2‐deoxyglucose to 2‐deoxyglucose phosphate was calculated by dividing the initial rate of 2‐deoxyglucose phosphate production by the 2‐deoxyglucose content in brain. The rate constant for the reverse reaction from 2‐deoxyyglucose phosphate to 2‐deoxyglucose was calculated from the decay constant of 2‐deoxyglucose phosphate. The rate constants estimated were 10.1 ± 1.4%/min (SD) and 3.00 ± 0.01%/min (SD), respectively, for the forward and reverse reactions. In the intravenous experiment, rate constants for both reactions were estimated by compartmental analysis. By fitting data to program SAAM‐27, the rate constants for the forward and reverse reactions were estimated as 11.4 ± 0.4%/min (SD) and 5.1 ± 0.4%/min (SD), respectively. The rate constants determined were compared to those for the reactions between glucose and glucose‐6‐phosphate, estimated previously from labeled glucoses. It is concluded that the rate of glucose utilization measured by the 2‐deoxyglucose method reflects the rate of the hexokinase reaction and not the rate of glucose utilization or brain energy utilization.
Endogenous Inhibitor of Ligand Binding to the Muscarinic Acetylcholine Receptor
Authors: Diaz‐Arrastia Ramon , Ashizawa Tetsuo , Appel Stanley H.
Published: October 2006

Abstract: An endogenous inhibitor of L‐[3H]quinuclinidinyl benzilate binding to the brain muscarinic acetylcholine receptor was identified. [3H]Quinuclinidinyl benzilate binding to rat brain synaptosomes was measured using a filtration assay. The inhibitor was prepared from several calf tissues and was found in highest specific activity in thymus. The loss of binding activity was slow, requiring a 30–40 min preincubation of the synaptosomes with the inhibitor, and reversed by removing the inhibitor by washing the membranes. Scatchard analysis of the binding data showed that the inhibition was noncompetitive resulting from both a decrease in affinity and a decrease in the number of binding sites. Zn2+ was required in low concentrations for this effect. Muscarinic acetylcholine receptor in synaptic membranes and in membranes free of most peripheral membrane proteins was still sensitive to inhibition. Preliminary characterization of the inhibitory molecule showed that it is of low molecular weight, moderately heat‐stable, and acidic. The inhibitor was inactivated by reagents that are nonspecific for nucleophiles, but not by reagents specific for primary amine or thiol groups.
True Renin in Human Pituitary Tissue
Authors: Mizuno Kenji , Ojima Motoko , Gotoh Mitsuhiro , Hashimoto Shigeatsu , Fukuchi Soitsu
Published: October 2006

Abstract: Readily detectable levels of renin activity were demonstrated in human pituitary tissues. This activity was inhibited by specific antibody raised against human renal renin, indicating that it was not due to the nonspecific action of proteases. It shared some biochemical features with well‐known kidney renin, such as molecular weight, optimum pH, and the presence of trypsin‐activatable inactive renin. These results suggest that true renin exists in human pituitary tissue.
Dephosphorylation of Microtubule‐Associated Protein 2, τ Factor, and Tubulin by Calcineurin
Authors: Goto Satoshi , Yamamoto Hideyuki , Fukunaga Kohji , Iwasa Takafumi , Matsukado Yasuhiko , Miyamoto Eishichi
Published: October 2006

Abstract: Calcineurin dephosphorylated microtubule‐associated protein 2 (MAP2) and τ factor phosphorylated by cyclic AMP‐dependent and Ca2+, calmodulin‐dependent protein kinases from the brain. Tubulin, only phosphorylated by the Ca2+, calmodulin‐dependent protein kinase, served as substrate for calcineurin. The concentrations of calmodulin required to give half‐maximal activation of calcineurin were 21 and 16 nM with MAP2 and τ factor as substrates, respectively. The Km and Vmaxvalues were in ranges of 1–3 μM and 0.4–1.7 μmol/mg/ min, respectively, for MAP2 and τ factor. The Km value for tubulin was in a similar range, but the Vmax value was lower. The peptide map analysis revealed that calcineurin dephosphorylated MAP2 and τ factor universally, but not in a site‐specific manner. The autophosphorylated Ca2+, calmodulin‐dependent protein kinase was not dephosphorylated by calcineurin. These results suggest that calcineurin plays an important role in the functions of microtubules via dephosphorylation.
k‐Binding and Degradation of [3H]Dynorphin A (1–8) and [3H]Dynorphin A (1–9) in Suspensions of Guinea Pig Brain Membranes
Authors: Gillan Maureen G. C. , Robson Linda E. , McKnight Alexander T. , Kosterlitz Hans W.
Published: October 2006

Abstract: Following incubation of [3H]dynorphin A (1–8) and [3H]dynorphin A (1–9) with suspensions of guinea pig brain membranes, analysis of the supernatants by HPLC has shown that both peptides are degraded at 25°C and at 0°C. Bestatin and captopril reduce degradation at 0°C but for a similar degree of protection at 25°C argininecontaining dipeptides are also required. The effects of these peptidase inhibitors on the degradation profiles indicate that [3H]dynorphin A (1–8) has three main sites of cleavage: the Tyr1‐Gly2, Arg6‐Arg7, and Leu5‐Arg6 bonds. With [3H]dynorphin A (1–9) as substrate the Arg7‐Ile8 and Ile8‐Arg9 bonds are also liable to cleavage. In binding assays, in contrast to the effects of peptidase inhibitors on the degradation of unbound [3H]dynorphin A (1–8) and [3H]dynorphin A (1–9), bestatin and captopril have little effect on the binding characteristics of the tritiated dynorphin A fragments at the k‐site at 0°C. However, at 25°C binding is low in the absence of peptidase inhibitors. When binding at μ‐ and δ‐sites is prevented, the maximal binding capacities of [3H]dynorphin A (1–8), [3H]dynorphin A (1–9), and [3H](–)‐bremazocine at the k‐site are similar; [3H]dynorphin A (1–9) has 5–10 times higher affinity for the k‐site than [3H]dynorphin A (1–8). Comparison of the effects of peptidase inhibitors on unbound dynorphin A fragments with their effects in binding assays suggests that the bound peptides are protected from the action of peptidases.
A Simple, Sensitive, and Economic Assay for Choline and Acetylcholine Using HPLC, an Enzyme Reactor, and an Electrochemical Detector
Authors: Damsma G. , Westerink B. H. C. , Horn A. S.
Published: October 2006

Abstract: A simple, efficient, economic, and sensitive method is presented for the detection of choline and acetylcholine in neuronal tissue using HPLC, a postcolumn enzyme reactor with immobilized enzyme, and electrochemical detection. The method is based on a separation of choline and acetylcholine by cation exchange HPLC followed by passage of the effluent through a postcolumn reactor containing a mixture of acetylcholinesterase and choline oxidase; the latter enzyme converts choline to betaine and hydrogen peroxide, the former enzyme hydrolyzes acetylcholine to acetate and choline. The hydrogen peroxide produced is electrochemically detected. A simple and efficient preparation of neuronal tissue is described using an optional prepurification step on Sephadex G‐10 columns, offering the possibility to detect choline and acetylcholine as well as catecholamines and their related metabolites in the same tissue sample. The sensitivity of the assay system is 250 fmol for choline and 500 fmol for acetylcholine.
Assay of Brain Calmodulin Levels Using High‐Performance Liquid Chromatography
Authors: Loullis Costas C. , Antonian Lida , Rauh Charles E. , Fliegner Karsten , Coupet Joseph , Lippa Arnold S.
Published: October 2006

Abstract: A simple, sensitive, and efficient HPLC method for the determination of calmodulin levels in brain tissue extracts is described. The assay is linear with respect to both calmodulin and protein concentrations. The specificity and validity of this assay for calmodulin is demonstrated by parallel radioimmunoassay determinations which give equivalent results. Determination of calmodulin levels in various brain regions revealed a high concentration of this protein in the hypothalamus, by comparison to other areas examined.
Modulation of [3H]Diazepam Binding in Rat Cortical Membranes by GABAA Agonists
Authors: Wong Erik H. F. , Iversen Leslie L.
Published: October 2006

Abstract: GABAA receptor agonists modulate [3H]diazepam binding in rat cortical membranes with different efficacies. At 23°C, the relative potencies for enhancement of [3H]diazepam binding by agonists parallel their potencies in inhibiting [3H]‐γ‐aminobutyric acid ([3H]GABA) binding. The agonist concentrations needed for enhancement of [3H]diazepam binding are up to 35 times higher than for [3H]GABA binding and correspond closely to the concentrations required for displacement of [3H]bicuculline methochloride (BMC) binding. The maximum enhancement of [3H]diazepam varied among agonists: muscimol = GABA > isoguvacine > 3‐aminopropane sulphonic acid (3APS) = imidazoleacetic acid (IAA) > 4,5,6,7‐tetrahydroisoxazolo (4,5,6)‐pyridin‐3‐ol (THIP) = taurine > piperidine 4‐sulphonic acid (P4S). At 37°C, the potencies of agonists remained unchanged, but isoguvacine, 3APS, and THIP acquired efficacies similar to GABA, whereas IAA, taurine, and P4S maintained their partial agonist profiles. At both temperatures the agonist‐induced enhancement of [3H]diazepam binding was reversible by bicuculline methobromide and by the steroid GABA antagonist RU 5135. These results stress the importance of studying receptor‐receptor in teraction under near‐physiological conditions and offer an in vitro assay that may predict the agonist status of putative GABAA receptor ligands.
Decreased Uptake and Release of D‐Aspartate in the Guinea Pig Spinal Cord After Partial Cordotomy
Authors: Potashner S. J. , Tran P. L.
Published: October 2006

Abstract: This study attempts to determine if L‐glutamate and/or L‐aspartate may be transmitters of neural tracts descending from the brain to the spinal cord. The uptake and electrically evoked release of D‐[3H]aspartate, a putative marker for L‐glutamate and L‐aspartate, were measured in the cervical enlargement of the guinea pig spinal cord. These activities were compared using unlesioned animals and others with a lesion on the right side of the spinal cord. Partial cordotomy (segment C5) produced a heavy loss of descending fibers, a small loss of primary sensory fibers, and a depression of the uptake and the Ca2+‐dependent, electrically evoked release of D‐aspartate ipsilateral and caudal to the lesion. Contralaterally, there was a moderate loss of corticospinal fibers, some loss of other descending axons, and a depression of D‐aspartate release. Dorsal rhizotomy (segments C4–T1) produced a heavy loss of primary sensory fibers ipsilateral to the lesion. Ipsilaterally, but not contralaterally, the uptake and release of D‐aspartate were depressed. Degeneration after partial cordotomy in combination with dorsal rhizotomy was assumed to be the sum of that produced by each lesion separately. This combined lesion depressed D‐aspartate uptake ipsilaterally and depressed D‐aspartate release on both sides of the cervical enlargement. None of the lesions altered the up‐take and the evoked release of [3H]GABA. These findings support the hypothesis that the synaptic endings of one or more neural tracts descending from the brain to the spinal cord mediate the uptake and release of D‐aspartate and, therefore, may use L‐glutamate or L‐aspartate as a transmitter.
Blood–Brain Barrier Transport of Valproic Acid
Authors: Cornford Eain M. , Diep Cynthia P. , Pardridge William M.
Published: October 2006

Abstract: Valproic acid distribution in brain is less than that of other anticonvulsants such as phenytoin or phenobarbital. Possible mechanisms for this decreased distribution space in brain include (a) increased plasma protein binding of valproate relative to the other anticonvulsants and (b) asymmetric blood‐brain barrier (BBB) transport of valproate such that the brain‐to‐blood flux exceeds the blood‐to‐brain flux. These mechanisms are investigated in the present studies using the intracarotid injection technique in rats and rabbits. In the rat, the brain uptake index (BUI) of [14C]valproate relative to [3H]water is 51 ± 6%, indicating the blood‐to‐brain transport of water is twofold greater than that of valproate. However, the BUI of [14C]valproate relative to [3H]water decreased with time after carotid injection during a 4‐min washout period, which indicates that brain‐to‐ßlood transport of valproate is greater than that of water. This suggests that the permeability of the BBB to valproate is polarized, with antiluminal permeability being much greater than luminal permeability. In rabbits, the BUI of [14C]valproate is 47 ± 7% in newborns and 17 ± 6% in adult animals. However, the high drug extraction in newborns may be attributed to decreased cerebral blood flow in the neonate as the BBB permeability‐surface area (PS) products are unchanged (e.g., PS= 0.13 and 0.11 ml min−1, g−1 in the newborn and adult rabbit, respectively). With regard to plasma protein binding effects on valproate transport, brain valproate uptake was also measured in the presence of human, lamb, pig, rat, horse, goat, hamster, dog, and mouse sera. Higher brain uptakes were observed when the unbound fraction of drug increased. However, our data indicate that a fraction of the valproic acid entering the capillaries bound to plasma proteins had the capacity to equilibrate with brain because of enhanced drug dissociation from albumin in the brain microcirculation. Since plasma protein‐bound valproate is available for uptake by brain, the major factor underlying the diminished distribution of the drug in brain appears to be the asymmetric transport properties of the BBB to valproic acid.
Changes in Histamine Metabolism in the Mouse Hypothalamus Induced by Acute Administration of Ethanol
Authors: Itoh Yoshinori , Nishibori Masahiro , Oishi Ryozo , Saeki Kiyomi
Published: October 2006

Abstract: The effect of acute ethanol administration on histamine (HA) dynamics was examined in the mouse hypothalamus. The steady‐state level of HA did not change after intraperitoneal administration of ethanol (0.5–5 g/kg), whereas the level of tele‐methylhistamine (t‐MH), a predominant metabolite of brain HA, increased when 3 and 5 g/kg of ethanol was given. Pargyline hydrochloride (80 mg/kg, i.p.) increased the level of t‐MH by 72.2% 90 min after the treatment. Ethanol at any dose given did not significantly affect the t‐MH level in the pargyline‐pretreated mice. Decrease in the t‐MH level induced by metoprine (10 mg/kg, i.p.), an inhibitor of HA‐N‐methyltransferase, was suppressed by ethanol (5 g/kg), thereby suggesting inhibition of the elimination of brain t‐MH. Ethanol (5 g/kg) significantly delayed the depletion of HA induced by (S)‐α‐fluoromethylhistidine (50 mg/kg, i.v.), a specific inhibitor of histidine decarboxylase. Therefore, a large dose of ethanol apparently decreases HA turnover in the mouse hypothalamus.
Cyclic AMP‐Dependent Protein Phosphorylation in Chemosensory Neurons: Identification of Cyclic Nucleotide‐Regulated Phosphoproteins in Olfactory Cilia
Authors: Heldman Judith , Lancet Doron
Published: October 2006

Abstract Chemosensory dendritic membranes (olfactory cilia) contain protein kinase activity that is stimulated by cyclic AMP and more efficiently by the nonhydrolyzable GTP analog guanosine‐5′‐O‐(3‐thio)triphosphate (GTPγS). In control nonsensory (respiratory) cilia, the cyclic AMP‐dependent protein kinase is practically GTPγS‐insensitive. GTPγS activation of the olfactory enzyme appears to be mediated by a stimulatory GTP‐binding protein (G‐pro‐tein) and adenylate cyclase previously shown to be enriched in the sensory membranes. Protein kinase C activity cannot be detected in the chemosensory cilia preparation under the conditions tested. Incubation of olfactory cilia with [γ‐32P]ATP leads to the incorporation of [32P]phosphate into many polypeptides, four of which undergo covalent modification in a cyclic nucleotide‐dependent manner. The phosphorylation of one polypeptide, pp24, is strongly and specifically enhanced by cyclic AMP at concentrations lower than 1 μM. This phosphoprotein is not present in respiratory cilia, but is seen also in membranes prepared from olfactory neuroepithelium after cilia removal. Cyclic AMP‐dependent protein kinase and phosphoprotein pp24 may be candidate components of the molecular machinery that transduces odor signals.
Potassium Ion Facilitation of Phosphoinositide Turnover Activation by Muscarinic Receptor Agonists in Rat Brain
Authors: Eva Carola , Costa Erminio
Published: October 2006

Abstract: In rat hippocampal slices kept in Krebs‐Henseleit medium, an increase of K+ ions to 12 mMpotentiates the stimulation of phosphoinositide turnover elicited by carbachol and (±)‐cis‐methyldioxolane. Oxotremorine is inactive if tested in Krebs‐Henseleit medium but it stimulates by 220% the phosphoinositide turnover when K+ is increased to 12 mM. The K+ facilitation of the carbachol stimulation of phosphoinositide turnover was blocked by pirenzepine, a muscarinic antagonist. This drug was equally potent in inhibiting the carbachol stimulation of phosphoinositide turnover both in normal and 12 mM K+ Krebs medium. This facilitatory effect of K+ appears to be preferential for muscarinic receptors, since it failed to increase the activation of phosphoinositide breakdown induced by norepinephrine and histamine. The K+ potentiation of the muscarinic stimulation of phosphoinositide turnover is not mediated by a release of one of the endogenous neurotransmitters stored in these slices because such a facilitation occurs in Ca2+ ‐deprived Krebs‐Henseleit medium and failed to occur following a depolarizing dose of veratrine. Our experiments excluded that K+ facilitates carbachol stimulation of phosphoinositide turnover because it modifies the binding characteristics of muscarinic receptors; however, they cannot exclude that K+ acts at the receptor transducer coupling.
Phylogenesis of Brain Glutamic Acid Decarboxylase from Vertebrates: Immunochemical Studies
Authors: Legay Francois , Pelhate Sylviane , Tappaz Marcel L.
Published: October 2006

Abstract: Brain high‐speed supernatants from various lower and higher vertebrates were subjected to sodium dodecyl sulfate (SDS)‐polyacrylamide gel electrophoresis, electroblot on nitrocellulose membranes, and immunolabelling using an anti‐glutamic acid decarboxylase (anti‐GAD) antiserum prepared from rat antigen. Rat brain extracts showed two distinct immunolabelled bands (MW 59,000 and 62,000 daltons). The molecular weight of the native enzyme was 120,000 daltons. The immunoblot pattern was not affected by a 3‐h incubation of the homogenate. In the substantia nigra, the decrease in the immunolabelling of both bands corresponded very closely to the decrease of GAD activity following lesioning of the striato‐nigral pathway. Moreover, experiments with preadsorbed antiserum showed that both subunits have common antigenic determinants. The immunolabelling was consistently more intense over the lightest band. The autoradiography of immunoprecipitated rat brain GAD. iodinated prior to electrophoresis, revealed two radiolabelled bands corresponding to the two immunolabelled ones. Their radioactivity was found in a one‐to‐five ratio which closely paralleled their respective immunolabelling intensity. Thus, the two subunits recognized by the antiserum are not present in stoichiometric proportions in the rat brain high‐speed supernatant. These findings suggest the existence of two homodimeric GAD with common antigenic determinants which are present in different amounts. Immunoprecipitation curves of brain GAD from rat, mouse, rabbit, monkey, human, quail, frog, and trout were similar, with a < 10‐fold maximum shift in affinity for GAD. GAD immunoblots from the various higher vertebrates showed a pattern similar to that obtained in rat. In contrast, in lower vertebrates (trout, frog) only one band was detected, the molecular weight of which was consistently found to be between the molecular weight values of the two bands immunodetected in the brains of higher vertebrates. The SDS‐electrophoresis of iodinated immunoprecipitated GAD from trout brain showed only one radiolabelled band which corresponded to the immunolabelled one. Native GAD from rat and trout brain exhibited an identical molecular weight (MW 120,000). These findings suggest that GAD exists in a unique homodimeric form in the brain of lower vertebrates. With antiserum preadsorbed on trout GAD, the immunolabelling of the two bands disappeared on rat brain immunoblots. All the antigenic determinants present on either subunit of the rat brain GAD are also present on the unique GAD subunit of the trout brain. Our results show that, although all immunogenic determinants have been highly conserved, a limited phylogenetic evolution on brain GAD has occurred among the vertebrates. This might have taken place at the posttranslational processing level, or alternatively at the genomic level, through, for example, gene duplication and subsequent mutations.
S100a0 (αα) Protein: Distribution in Muscle Tissues of Various Animals and Purification from Human Pectoral Muscle
Authors: Kato Kanefusa , Kimura Shigeki , Haimoto Hajime , Suzuki Fujiko
Published: October 2006

Abstract: When the concentrations of α‐S100 (α subunit of S100 protein) and β‐S100 (β subunit) proteins in various tissues of human and rat were determined by the immunoassay method, immunoreactive β‐S100 was present at high levels in the CNS, adipose tissue, and cartilaginous tissue. In contrast, the α‐S100 was found in the heart and skeletal muscles at concentrations much higher than in the CNS. The concentration of α‐S100 protein was also high in the heart and skeletal muscles of bovine, porcine, canine, and mouse. Since β‐S100 protein levels in those tissues were low, it was suggested that S100 protein in the muscle tissues is present mainly as the αα form (S100a0 protein). To confirm the above findings, immunoreactive α‐S100 protein was purified from human pectoral muscle by employing column chromatographies with butyl‐Sepharose, diethylaminoethyl (DEAE)‐Sepharose, Sephadex G‐75, and finally with an anion‐exchange Mono Q column in a HPLC system. The elution profile of α‐S100 protein from the Mono Q column suggested some heterogeneity of the final preparation. However, each of these fractions traveled with a single band at a position similar to that of bovine S100a0 protein on slab‐gel electrophoresis. The amino acid composition of the final preparation was very similar to the composition of bovine S100a0 protein. The purified α‐S100 protein was eluted from a gel‐filtration column (Superose 12) in the same fraction as bovine S100a0 protein. These results indicate that the α‐S100 protein purified from human pectoral muscle is S100a0 protein, and that S100a0 protein is present universally in the heart and skeletal muscles in a variety of animals at much higher levels than in the brain.
Metorphamide Levels in Chromaffin Cells Increase After Treatment with Reserpine
Authors: Eiden Lee E. , Zamir Nadav
Published: October 2006

Abstract: Exposure of bovine chromaffin cells in primary culture to 0.01–1 μM reserpine caused a dose‐ and time‐dependent increase in intracellular levels of the amidated enkephalin peptide metorphamide. Maximal levels (approximately 800% of control) were obtained at 0.1 μM reserpine and increased levels were apparent by 16 h of treatment. Metorphamide increases were at least fivefold more than that of either Met‐ or Leu‐enkephalin, suggesting that reserpine stimulates both enkephalin processing and amidation in the secretory vesicle. Treatment with elevated potassium, which increases enkephalin levels by stimulating production of preproenkephalin messenger RNA, elicited an increase in metorphamide levels equivalent to, but not greater than, the increase in Met‐enkephalin pentapeptide. The ratio of Met‐enkephalin to metorphamide in untreated chromaffin cells is approximately 140:1, whereas the final Met‐enkephalin: metorphamide ratio in reserpinized chromaffin cells is approximately 30:1, similar to the Met‐enkephalin:metorphamide ratio in enkephalinergic neurons of the CNS.
Circadian Rhythm in Pineal N‐Acetyltransferase Activity: Rapid Phase Reversal and Response to Shorter than 24‐Hour Cycles (IV)
Authors: Binkley Sue , Mosher Karen , White Beatrix H.
Published: October 2006

Abstract: N‐Acetyltransferase (NAT) is an enzyme whose rhythmic activity in the pineal gland and retina is responsible for circadian rhythms in melatonin. The NAT activity rhythm has circadian properties such as persistence in constant conditions and precise control by light and dark. Experiments are reported in which chicks (Callus domesticus), raised for 3 weeks in 12 h of light alternating with 12 h of dark (LD12:12), were exposed to 1–3 days of light‐dark treatments during which NAT activity was measured in their pineal glands, (a) In LD12:12, NAT activity rose from <4.5 nmol/pineal gland/h during the light‐time to 25–50 nmol/pineal gland/h in the dark‐time. Constant light (LL) attenuated the amplitude of the NAT activity rhythm to 26‐ 45% of the NAT activity cycle in LD 12:12 during the first 24 h. (b) The timing of the increase in NAT activity was reset by the first full LD12:12 cycle following a 12‐h phase shift of the LD12:12 cycle (a procedure that reversed the times of light and dark by imposition of either 24 h of light or dark). This result satisfies one of the criteria for NAT to be considered part of a circadian driving oscillator, (c) In <24‐h cycles [2 h of light in alternation with 2 h of dark (LD2:2), 4 h of light in alternation with 4 h of dark (LD4: 4), and 6 h of light in alternation with 6 h of dark (LD6:6)], NAT activity rose in the dark during the chicks’ previously scheduled dark‐time but not the previously scheduled light‐ time of LD 12:12. In a cycle where 8 h of light alternated with 8 h of dark (LD8:8), NAT activity rose in both 8‐h dark periods, even though the second one fell in the light‐time of the prior LD12:12 schedule. The results support a prior hypothesis that divides the underlying system controlling NAT activity into a sensitive period and a refractory period.
Molecular Polymorphism of Head Acetylcholinesterase from Adult Houseflies (Musca domestica L.)
Authors: Fournier D. , Cuany A. , Bride J. M. , Bergé J. B.
Published: October 2006

Abstract: Acetylcholinesterase (AChE) from housefly heads was purified by affinity chromatography. Three different native forms were separated by electrophoresis on poly‐acrylamide gradient gels; Two hydrophilic forms presented apparent molecular weights of 75,000 (AChE1) and 150,000 (AChE2). A third component (AChE3) had a migration that depended on the nature and concentration of detergents. In the presence of sodium deoxycholate in the gel, AChE3 showed an apparent molecular weight very close to that of AChE2. Among the three forms, AChE3 was the only one found in purified membranes. The relationships among the various forms were investigated using reduction with 2‐mercaptoethanol or proteolytic treatments. Such digestion converted purified AChE3 into AChE2 and AChE1, and reduction of AChE3 and AChE2 by 2‐mercaptoethanol gave AChE1, in both cases with a significant loss of activity. These data indicate that the three forms of purified AChE may be classified as an active hydrophilic monomeric unit (G1) plus hydrophilic and amphiphilic dimers. These two components were termed G2sand G2m, where “s” refers to soluble and “m” to membrane bound.).
Immunohistochemistry and Biosynthesis of N‐Acetylaspartylglutamate in Spinal Sensory Ganglia
Authors: Cangro C. B. , Namboodiri M. A. A. , Sklar L. A. , Corigliano‐Murphy A. , Neale J. H.
Published: October 2006

Abstract: N‐Acetylaspartylglutamate (NAAG) is a nervous system‐specific dipeptide which has been implicated in chemical neurotransmission. Antisera were prepared against NAAG in order to study its cellular distribution. When these antisera were applied to tissue sections of rat spinal sensory ganglia, NAAG‐like immunoreactivity was detected within a subpopulation of relatively large neuronal cell bodies in cervical, lumbar, and thoracic ganglia. In order to confirm the presence of NAAG within these neurons, the dipeptide was extracted and purified from spinal ganglia using high‐performance liquid chromatography and its composition confirmed by amino acid analysis. Further, the biosynthesis of NAAG was studied in vitro by following the incorporation of either [3H]glutamine or [3H]glutamate into the glutamate residue of the purified dipeptide. [3H]Aspartate was not incorporated efficiently into NAAG under these conditions, suggesting a precursor role for the large N‐acetylaspartate pool. The incorporation of radiolabeled amino acids into newly synthesized NAAG by spinal sensory ganglia was not inhibited by incubation of the cells with anisomycin or cycloheximide at concentrations which significantly inhibited protein synthesis. These data suggest that NAAG is present in a subpopulation of primary afferent spinal neurons and that its biosynthesis is mediated by a dipeptide synthetase.