bluuebunny/crossref_metadata_2025 huggingface dataset:
The Stability of Solubilized Mammalian Muscle Acetylcholine Receptors During Purification by Monoclonal Immunoadsorption
Authors: Momoi Mariko Yoshida , Lennon Vanda A.
Published: October 2006

Abstract: The stability of nicotinic acetylcholine receptors (AChR) solubilized from mammalian skeletal muscle in nonionic detergent was investigated under various conditions of pH, chaotropic ions, and unfolding reagents in order to allow its purification in high yield by immunoadsorption to monoclonal antibodies. Preservation of the antigenicity and/or binding sites for α‐bungarotoxin was used as an indicator of the receptor protein's integrity. Both were preserved in the pH range 6.5–8.0, but when exposed for 1 h at 4°C to a pH outside this range, >50% activity was lost. Of the chaotropic ions studied (NaSCN, Nal, NaNO3, NaCl), only NaCl was tolerated. Most of the AChR's toxin‐binding activity was preserved after ex posure to 2 M NaCl, which was suitable for dissociating AChR when a monoclonal antibody with relatively low binding affinity was selected as the immunoadsorbent. Yields of purified AChR were optimal (30%) when a low amount of monoclonal antibody was coupled to cyanogen bromide‐activated agarose (1 mg protein/ml gel).
Inhibition of Rat Brain Microsomal Na+,K+‐ATPase by S‐Adenosylmethionine
Authors: Hattori Hiroshi , Kanfer Julian N.
Published: October 2006

Abstract: Rat brain microsomes were preincubated with S‐adenosylmethionine (SAM), MgCl2, and CaCl2, then re‐isolated, and the activity of Na+,K+‐ATPase determined. SAM inhibited the Na+,K+‐ATPase activity compared with microsomes subjected to similar treatment in the absence of SAM. A biphasic inhibitory effect was observed with a 50% decrease at a SAM concentration range of 0.4 μM‐3.2 μM and a 70% reduction at a concentration range above 100 μM. Inclusion of either S‐adenosylhomocysteine or 3‐deazaadenosine in the preincubations prevented the SAM inhibition of Na+,K+‐ATPase activity. The inhibition by SAM appeared to be Mg2+‐ or Ca2+‐dependent.
Various Types of Thyrotropin‐Releasing Hormone Receptors in Discrete Brain Regions and the Pituitary of the Rat
Authors: Funatsu Kunihiko , Teshima Shigeki , Inanaga Kazutoyo
Published: October 2006

Abstract: Receptors for thyrotropin‐releasing hormone (TRH) in the rat brain and the pituitary are heterogenous. The receptors were classified into four types according to the dissociation constant (KD). High‐affinity receptors (KD < 3 nM) are present in the pituitary, hypothalamus, amygdala, and limbic forebrain which contains the nucleus accumbens and the septum. Intermediate‐affinity receptors (KD, 5–16 nM) are evidently present in the frontal cortex, hippocampus, striatum, thalamus, and the brainstem, but may also be present in other regions. Lowaffinity TRH receptors (KD, 50–80 nM) are seen in the limbic forebrain, amygdala, and the hypothalamus. Verylow‐affinity receptors (KD, 215 nM) exist in the pituitary. Experiments using DN‐1417 (γ‐butyrolactone‐γ‐carbonyl‐histidyl‐prolinamide citrate), a synthetic TRH analogue with a more potent central activity, indicated the presence of TRH receptors having a high affinity to DN‐1417 at least in the limbic forebrain but not in the pituitary. This type of receptor is not labeled by [3H](3methyl‐histidine2)‐TRH. Density of the TRH receptor is the highest in the pituitary and next highest in the amygdala.
Decreased Incorporation of [3H]Inositol and [3H]Glycerol into Glycerolipids of Sciatic Nerve from the Streptozotocin Diabetic Rat
Authors: Bell Margaret E. , Eichberg Joseph
Published: October 2006

Abstract: The incorporation of [3H]myo‐inositol into individual phosphoinositides and of [3H]glycerol into glycerolipids was determined in sciatic nerve obtained from normal and streptozotocin diabetic rats and incubated in vitro. The uptake of inositol into lipid was approximately linear with time. More than 80% of the label was present in phosphatidylinositol with the remainder divided about equally between phosphatidylinositol phosphate and phosphatidylinositol‐4,5‐bisphosphate. Labeling was unchanged 2 weeks after induction of diabetes, but was reduced by 32% after 20 weeks of the disease. Glycerol incorporation occurred primarily into phosphatidylcholine and triacylglycerol and was depressed up to 45% into major phosphoglycerides in nerves from both 2‐ and 20‐week diabetic animals. Triacylglycerol labeling was also substantially decreased, and the reduction was comparable in intact and epineurium free nerve, suggesting that a metabolically active pool of this compound, which is sensitive to hyperglycemia and/or insulin deficiency, is located in or immediately adjacent to the nerve fibers. The considerable decline in incorporation of these lipid precursors in diabetic nerve may be related to impaired inositol transport and to decreased overall energy utilization by the tissue.
Cholesterol Biosynthesis and Its Regulation in Dissociated Cell Cultures of Fetal Rat Brain: Developmental Changes and the Role of 3‐Hydroxy‐3‐Methylglutaryl Coenzyme A Reductase
Authors: Volpe Joseph J. , Goldberg Richard I. , Bhat Narayan R.
Published: October 2006

Abstract: Primary cultures of cells dissociated from fetal rat brain were utilized to define the developmental changes in cholesterol biosynthesis and the role of 3‐hydroxy‐3‐methylglutaryl coenzyme A (HMG‐CoA) reductase in the regulation of these changes. Cerebral hemispheres of fetal rats of 15–16 days of gestation were dissociated mechanically into single cells and grown in the surface‐adhering system. Cholesterol biosynthesis, studied as the rate of incorporation of [14C]acetate into digitonin‐precipitable sterols, was shown to exhibit two distinct increases in synthetic rates, a prominent increase after 6 days in culture and a smaller one after 14 days in culture. Parallel measurements of HMG‐CoA reductase activity also demonstrated two discrete increases in enzymatic activity, and the quantitative and temporal aspects of these increases were virtually identical to those for cholesterol synthesis. These data indicate that cholesterol biosynthesis undergoes prominent alterations with maturation and suggest that these alterations are mediated by changes in HMG‐CoA reductase activity. The timing of the initial prominent peak in both cholesterol biosynthesis and HMG‐CoA reductase activity at 6 days was found to be the same as the timing of the peak in DNA synthesis, determined as the rate of incorporation of [3H]thymidine into DNA. The second, smaller peak in reductase activity and sterol biosynthesis at 14 days occurred at the time of the most rapid rise in activity of the oligodendroglial enzyme, 2′:3′‐cyclic nucleotide 3′‐phosphohydrolase (CNP). These latter observations suggest an intimate relationship of the sterol biosynthetic pathway with cellular proliferation and with oligodendroglial differentiation in developing mammalian brain.
Comparison of the Vascular Permeability of the Brain and the Spinal Cord to Mannitol and Inulin in Rats
Authors: Daniel P. M. , Lam D. K. C. , Pratt O. E.
Published: October 2006

Abstract: The kinetics of the movement of [14C]mannitol and [14C]inulin from the blood into the CNS of the rat were measured. The spinal cord was found to have a higher permeability to these two substances than the brain. It is likely that the channels through which the tracers diffused are larger in the capillaries of the spinal cord than those in the brain.
Synthesis of the Novel Dipeptide β‐Aspartylglycine by Aplysia Ganglia
Authors: McCaman Marilyn W. , Stetzler Judith
Published: October 2006

Abstract: Isolated ganglia from Aplysia californica rapidly took up [14Clglycine or [14C]aspartate from a seawater medium. Approximately 20% of the tissue radioactivity was recovered in the peptides β‐aspartylglycine and glutathione after incubation with [14C]glycine. Compared with other individual cells isolated from the abdominal ganglion, the glycine‐containing white cells (R3‐R14) incorporated less [14C]glycine into β‐aspartylglycine, but similar amounts into glutathione. In contrast, [14C]aspartate was metabolized primarily to nonamino dicarboxylic acids and relatively little radioactivity was incorporated into β‐aspartylglycine.
Stimulation of Calcium Uptake in PC12 Cells by the Dihydropyridine Agonist BAY K 8644
Authors: Greenberg David A. , Carpenter Celia L. , Cooper Edward C.
Published: October 2006

Abstract: Methyl 1,4‐dihydro‐2,6‐dimethyl‐3‐nitro‐4‐(2‐trifluoromethylphenyl)‐pyridine‐5‐carboxylate (BAY K 8644), an analog of dihydropyridine calcium channel antagonists, stimulated 45Ca uptake into PC12 pheochromocytoma cells. Half‐maximal stimulation occurred at 80 nM BAY K 8644. Enhancement of uptake was inhibited by cationic and organic calcium channel blockers, but not by tetrodotoxin, which is consistent with an effect on voltage‐dependent calcium channels. Stimulation of 45Ca uptake by BAY K 8644 occurred only at elevated concentrations of extracellular K+, suggesting that BAY K 8644 may interact with calcium channels in the open (activated) state.
Phospholipid Methylase Activity, [3H]S‐Adenosyl‐l‐Homocysteine Binding, and S‐Adenosyl‐l‐Methionine and S‐Adenosyl‐l‐Homocysteine Levels in Rat Brain During Maturation
Authors: Gharib A. , Rey C. , Fonlupt P. , Sarda N. , Pacheco H.
Published: October 2006

Abstract: The changes in activity of phospholipid methyltransferase I and [3H]S‐adenosyl‐l‐homocysteine ([3H]SAH) binding were determined in cortical membrane preparations from newborn rats and rats 1, 2, and 8 months old. The activity of phospholipid methyltransferase I and the [3H]SAH binding were significantly greater (respectively, +30 and +40%) in newborn rats than in 1‐, 2‐, and 8‐month‐old rats. The methylated products at days 1 and 30 were identical. These changes in methyltransferase activity may be correlated with variations in concentration of S‐adenosyl‐l‐methionine (SAM) and SAH. The endogenous SAM level was higher and the SAH level was lower in newborn compared with adult rats. These data suggested that the processes of methylation were favored in newborn rats. The modifications observed after treatment with l‐homocysteine reinforced this hypothesis.
Cholinesterases in Single Nerve Cells Isolated from the Locus Ceruleus and from Nucleus of the Facial Nerve of the Rat: A Microgasometric Study
Authors: Sket Dusan , Pavlin Rudi
Published: October 2006

Abstract: Cholinesterase activity in single nerve cell bodies isolated from the locus ceruleus and nucleus of the facial nerve of the rat was analyzed by the microgasometric method. Acetylcholinesterase activity is about the same in both types of cells. Nonspecific cholinesterase is present in noradrenergic cells of the locus ceruleus but not in the cholinergic cells of the nucleus of the facial nerve. The total activity of cholinesterases and the activity of acetylcholinesterase in nerve cell bodies isolated from the locus ceruleus remains practically unchanged from the tenth postnatal day until the age of 24 months. Depletion of noradrenaline by a high dose of reserpine does not influence the total activity of cholinesterases in nerve cell bodies of locus ceruleus.
Degradation of Rat Brain Cholinergic Muscarinic Receptors In Vitro: Enhancement by Agonists and Inhibition by Antagonists
Authors: Roskoski Robert , Guthrie Richard , Roskoski Laura M. , Rossowski Wojciech
Published: October 2006

Abstract: Cholinergic muscarinic receptors undergo proteolytic degradation in vitro under physiological conditions as shown by a loss in [3H]quinuclidinylbenzilate binding activity. The serine protease inhibitor phenyl‐methylsulfonyl fluoride was very effective in diminishing the receptor loss. Soybean trypsin inhibitor was less effective. Both EDTA and EGTA were also effective in abolishing receptor degradation, suggesting the involvement of metallopeptidases in the process. Calcium‐dependent neutral proteases requiring sulfhydryl reducing agents did not seem to be involved in receptor degradation. Dithiothreitol failed to enhance receptor degradation and iodoacetamide, leupeptin, and antipain, inhibitors of this enzyme class, failed to alter receptor loss as measured by radioligand binding. Most of the proteolytic activity occurred in the cytosol and was readily resolved from the receptor in the membrane fraction. We found that [3H]quinuclidinylbenzilate, an antagonist, inhibited the rate of receptor loss. On the other hand, agonists (acetylcholine, methacholine, and muscarine) appeared to enhance the rate of receptor loss. We postulate that these opposite effects are due to differences in receptor conformation in response to ligand binding. Susceptibility to proteolysis may therefore serve as a probe for receptor conformation.
Phosphorylation of the Postsynaptic Density Glycoprotein gp 180 by Ca2+/Calmodulin‐Dependent Protein Kinase
Authors: Gurd James W.
Published: October 2006

Abstract: Postsynaptic densities (PSDs) were prepared by the aqueous two‐phase extraction of synaptic membranes in the presence of n‐octyl glucoside. Incubation of postsynaptic densities with [γ‐32P]ATP resulted in the incorporation of 32P into a range of proteins. Isolation of glycoproteins from 32P‐labelled PSDs by affinity chromatography on concanavalin A‐agarose identified the postsynaptic glycoprotein of apparent M, 180,000 (gp180) as a substrate for endogenous protein kinase(s). When the phosphorylation reaction was performed in the presence of Ca2+ and calmodulin, there was an overall 13‐ fold increase in the phosphorylation of PSD proteins. The largest effects of calmodulin were associated with two proteins of molecular weights 51,000 and 60,000, which showed average calmodulin‐dependent increases in phosphorylation of 68‐fold. The phosphorylation of gp180 was increased 7.5‐fold in the presence of calmodulin. Fifty percent of maximum phosphorylation of proteins and glycoproteins occurred with a free Ca2+ concentration of 0.3 × 10−6M. The amounts 12.6 μg/ml and 9.1 μg/ml of calmodulin were required for 50% of maximum phosphorylation of proteins and glycoproteins, respectively. Peptide mapping experiments identified three major phosphorylation sites in gp180. The phosphorylation of all three sites was increased in the presence of calmodulin. Phosphoamino acid analysis of gp180 revealed that [32P]phosphoserine and [32P]phosphothreonine were both produced during the phosphorylation reaction, with phosphoserine being the predominant product. The phosphorylation of both amino acids was increased in the presence of calmodulin. [32P]phosphotyrosine was also identified as a product of the phosphorylation of gp180.
Effects of Monovalent Ions on the Transport of Noradrenaline Across the Plasma Membrane of Neuronal Cells (PC‐12 Cells)
Authors: Harder R. , Bönisch H.
Published: October 2006

Abstract: The dependence on Na+, K+, and C1− of uptake and accumulation of [3H]noradrenaline was studied in plasma membrane vesicles isolated from PC‐12 pheochromocytoma cells. Plasma membrane vesicles accumulated [3H]noradrenaline when an inward‐directed gradient for Na+ and an outward‐directed gradient for K+ were imposed across the vesicle membrane. Under these conditions, initial rates of uptake of [3H]noradrenaline were saturable (Km= 0.14 μM) and inhibited by a series of substrates and inhibitors of “uptake1.’ The IC50 values were positively correlated with those for inhibition of uptake into intact PC‐12 cells. Uptake and accumulation [3H]noradrenaline in plasma membrane vesicles were absolutely dependent on external Na+ and C1−; they were dependent on an inwardly directed gradient for Na+ but less dependent on an inwardly directed gradient for C1−. Internal K+ strongly enhanced uptake and accumulation of [3H]noradrenaline. Rb+, but not Li+, had the capacity to replace internal K+. Two explanations are proposed for this effect of internal K+: (a) creation of a K+ diffusion potential (inside negative) provides a driving force for inward transport, and/or (b) K+ increases the turnover rate by formation of a highly mobile potassium–carrier complex. A hypothetical scheme for the transport of noradrenaline is presented.
Relationship Among Calmodulin‐, Forskolin‐, and Guanine Nucleotide‐Dependent Adenylate Cyclase Activities in Cerebellar Membranes: Studies by Limited Proteolysis
Authors: Malnoë Armand , Cox Jos A.
Published: October 2006

Abstract: Adenylate cyclase activity in bovine cerebellar membranes is regulated by calmodulin, forskolin, and both stimulatory (Ns) and inhibitory (Ni) guanine nucleotide‐binding components. The susceptibility of the enzyme to chymotrypsin proteolysis was used as a probe of structure‐function relationships for these different regulatory pathways. Pretreatment of membranes with low concentrations of chymotrypsin (1–2 μg/ml) caused a three‐ to fourfold increase in basal adenylate cyclase activity and abolished the Ca2+‐dependent activation of the enzyme by calmodulin. In contrast, the stimulation of the enzyme by GTP plus isoproterenol was strongly potentiated after protease treatment, an effect that mimics the synergistic activation of adenylate cyclase by Ns and calmodulin in unproteolyzed membranes. Limited proteolysis revealed low‐ and high‐affinity components in the activation of adenylate cyclase by forskolin. The lowaffinity component was readily lost on proteolysis, together with calmodulin stimulation of the enzyme. The activation via the high‐affinity component was resistant to proteolysis and nonadditive with the Ns‐mediated activation of the enzyme, suggesting that both effectors utilize a common pathway. The inhibitory effect of low concentrations (10−7M) of guanyl‐5′‐yl imidodiphosphate [Gpp(NH)p] on forskolin‐activated adenylate cyclase was retained after limited proteolysis of the membranes, indicating that the proteolytic activation does not result from an impairment of the Ni subunit. Moreover, in the rat cerebellum, proteolysis as well as calmodulin was found to enhance strongly the inhibitory effect of Gpp(NH)p on basal adenylate cyclase activity. Our results suggest that calmodulin and Ns/Ni interact with two structurally distinct but allosterically linked domains of the enzyme. Both domains appear to be involved in the mode of action of forskolin.
Distribution of Nsp100 and Nsp100 Kinase, a Nerve Growth Factor–Sensitive Phosphorylation System, in Rat Tissues
Authors: Hama Tokiko , Guroff Gordon
Published: October 2006

Abstract: Previous work from this laboratory has shown that in PC12 cells the phosphorylation of a specific soluble protein is decreased by nerve growth factor treatment. The protein, designated Nsp100, and its kinase have been separated and partially purified from PC12 cells. In the present work, the tissue distribution of Nsp100 phosphorylation in 5‐day‐old‐and adult rats was studied. In adult rats, phosphorylation of an Nsp100‐like protein was observed in brain, adrenal gland, testis, and muscle, but not in liver or kidney. In 5‐day‐old rats, a similar phosphorylation was observed in brain, adrenal gland, superior cervical ganglia, liver, spleen, kidney, and muscle. In PC12 cells, Nsp100 phosphorylation is completely inhibited by 5 × 10−5M Zn2+ and is completely inactivated by treatment at 50°C for 2 min. The phosphorylation of the Nsp100‐like protein in both adult and 5‐day‐old rats showed the same characteristics. Partial purification of Nsp100 and Nsp100 kinase from the brains of 5‐day‐old rats was carried out using the procedures developed for PC12 cells. Nsp100 and Nsp100 kinase were separated on diethylaminoethyl‐Sephacel, and the kinase was eluted with 0.3 M NaCl; the same results have previously been obtained with PC12 cells. Phosphorylated Nsp100 from brain and from PC12 cells was compared by proteolysis on sodium dodecyl sulfate gels; similar peptide patterns were generated from the two samples. The phosphorylation of the Nsp100‐like protein by extracts of superior cervical ganglia was decreased by prior in vitro treatment of the ganglia with nerve growth factor, showing that the effect of nerve growth factor on Nsp100 phosphorylation observed in PC12 cells also occurs in normal target tissues.
Alterations of the Eicosanoid Synthetic Capacity of Rat Brain Microvessels Following Ischemia: Relevance to Ischemic Brain Edema
Authors: Koide Tohru , Gotoh Osamu , Asano Takao , Takakura Kintomo
Published: October 2006

To know the mechanism underlying ischemic brain edema, a time‐course analysis of the eicosanoid synthetic capacity of brain microvessels was carried out using unilateral, middle cerebral artery (MCA)‐occluded rats. Concomitant with the development of brain edema the synthetic capacity of all products, including cyclooxygenase and lipoxygenase products, increased significantly. Next the effects of 15‐hydroperoxyarachidonic acid (15‐HPAA) on the synthetic capacity of microvessels were examined. The drug caused a generalized increase of each product, the profile of which was similar to that obtained with ischemic hemispheres, although the ratios of each product differed somewhat among them. The enhanced synthesis of eicosanoids by 15‐HPAA was markedly suppressed by radical scavengers such as α‐tocopherol, hydroquinone, and 1,2‐bis(nicotineamide)‐propane. Furthermore, the evolution of brain edema was virtually suppressed by the systemic administration of 1,2‐bis(nicotineamide)‐propane. The above result suggests that the enzyme activity of the arachidonic acid (AA) cascade of microvessels is stimulated by its own products. Such a mechanism will form a vicious cycle that accelerates the accumulation of free radicals within microvessels and thus may play a role in the progressing disruption of the blood‐brain barrier (BBB) following ischemia.
Two‐Step Immunoaffinity Purification of Acetylcholinesterase from Rabbit Brain
Authors: Mintz K. P. , Brimijoin S.
Published: October 2006

Acetylcholinesterase (AChE; EC 3.1.1.7) extracted in 1% Triton X‐100 from rabbit brain was purified 2,000‐fold by chromatography on agarose conjugated with a monoclonal antibody directed against human red blood cell cholinesterase. After elution from the immunoadsorbent with pH 11 buffer, the preparation was purified further by affinity chromatography on phenyltrimethylammonium‐Sepharose 4B with decamethonium elution. Overall yield of purified enzyme was 37% of the AChE originally solubilized, with a specific activity of 2,950 units/mg protein. Electrophoresis under reducing conditions in 7.5% sodium dodecyl sulfate polyacrylamide gels revealed only one silver‐staining polypeptide band. A streamlined purification procedure enabled the isolation of electrophoretically homogenous AChE to be completed in fewer than 7 days, at yields exceeding 50%. Electrophoretic analysis of purified AChE indicated an apparent MW of 71,000 for the monomeric subunit. Gel filtration and sucrose density gradient centrifugation in the presence of Triton X‐100 showed little difference between the properties of the native and the purified enzyme. The molecular mass of the main species was estimated from the gel filtration and sedimentation data to be 280,000 daltons. Kinetic parameters of the purified protein (Km= 0.16 ± 0.01 mM) were close to those of the native enzyme (Km= 0.12 ± 0.01 mM) when examined with acetylthiocholine iodide as substrate. The two‐step immunopurification procedure presented in this communication offers a convenient route to homogenous neural AChE in quantities useful for detailed biochemical and immunochemical study.
Allosteric Modulation of Flunitrazepam Binding to Rat Brain Benzodiazepine Receptors by Methyl β‐Carboline‐3‐Carboxylate
Authors: Chiu Ted H. , Rosenberg Howard C.
Published: October 2006

The inhibition of flunitrazepam (FNP) binding to rat brain benzodiazepine (BZ) receptors by methyl β‐carboline‐3‐carboxylate (MCC) was studied. Biphasic dissociation was observed for [3H]FNP and [3H]MCC in cerebral cortex, cerebellum, and hippocampus, although the dissociation of [3H]MCC was much faster. The dissociation rate of [3H]FNP was increased by MCC in the cerebellum, but was not altered in cerebral cortex or hippocampus. [3H]FNP binding stimulated by γ‐aminobutyric acid was enhanced in the presence of MCC in all three regions examined. These results indicate that MCC exerts these effects by interacting with allosteric sites that are different from the FNP recognition sites on the BZ receptors.
Separation of Neuropeptides by HPLC: Evaluation of Different Supports, with Analytical and Preparative Applications to Human and Porcine Neurophysins, β‐Lipotropin, Adrenocorticotropic Hormone, and β‐Endorphin
Authors: Richter W. O. , Schwandt P.
Published: October 2006

The separation of 38 hypophyseal or hypothalamic peptides and proteins by reversed‐phase HPLC is described. The efficacy of several supports is discussed: large‐pore reversed‐phase silica supports turned out to be most effective in the separation of neuropeptides and proteins. The use of high sodium phosphate and phosphoric acid in the eluent resulted in high yields and better separation. On the basis of this procedure, β‐lipotropin, β‐endorphin, and adrenocorticotropic hormone were prepared from porcine pituitary glands, and human neurophysins from human pituitary posterior lobes. Moreover, this system is used for the analysis of the perifusate of pituitary posterior lobes, showing the release of microheterogeneous neurophysins.
Halothane‐Induced Alterations of Glucose and Pyruvate Metabolism in Rat Cerebra Synaptosomes
Authors: Johnson G. V. W. , Hartzell Charles R.
Published: October 2006

Synaptosomes isolated from rat cerebra were used to study the effects of the inhalational anesthetic, halothane, on cholinergic processes. To identify possible mechanisms responsible for the depression of acetylcholine synthesis, we examined the effects of halothane on precursor metabolite metabolism involved with supplying the cytosol with acetyl‐CoA for acetylcholine synthesis. Three percent halothane/air (vol/vol) depressed 14CO2 evolution from labeled pyruvate and glucose. Steadystate 14CO2 evolution from [1‐14C]glucose was depressed 84% by halothane, while 14CO2 evolution from [6‐14C]glucose and [3,4‐14C]glucose was decreased 67 and 52%, respectively, when compared with control conditions. Halothane inhibited the activities of both pyruvate dehydrogenase (14% depression) and ATP‐citrate lyase (32% depression). Total synaptosomal acetyl‐CoA concentrations were unaffected by halothane. Three percent halothane/air (vol/vol) caused a 77% increase in medium glucose depletion rate from 1.38 nmol (mg protein)−1 min−1 to 2.44 nmol (mg protein)−1 min−1. Production of lactate by the synaptosomes in the presence of halothane increased by 231% from a control rate of 1.44 nmol (mg protein)−1 min−1 to 4.77 nmol (mg protein)−1 min−1. Lactate production rate from pyruvate was also enhanced by 56% in the presence of halothane. These data lend support to the concept that the NAD+/NADH potential may be involved in the halothane‐induced depression of acetylcholine synthesis.
Composite Technique for Regional Neurochemical Studies: Measurement of Energy and Neurotransmitter Metabolites in Single Tissue Sample
Authors: Djuricic Bogdan M. , Ueki Yasuyuki , Spatz Maria
Published: October 2006

A combined method is described for the determination of various metabolites from a single tissue sample of the brain. It comprises a quick inactivation of cerebral enzymes by microwave irradiation, easy separation of the desired brain regions, and perchloric acid extraction of tissue substances, which are assayed either by specific enzymatic techniques or by HPLC with electrochemical detection. The obtained values of most energy and neurotransmitter metabolites in the brain are in agreement with those reported using other methods. However, this technique, in contrast to the brain freezing in vitro or freeze‐blowing, provides a more efficient procedure for rapid arrest of cerebral metabolism even in the deep brain structures and is therefore suitable for detection of early changes particularly those occurring in experimental pathological conditions such as ischemia.
Effects of High‐Pressure Helium on γ‐[3H]Aminobutyric Acid Release from the Isolated Frog Spinal Cord
Authors: Bichard A. R. , Little H. J.
Published: October 2006

Abstract: The application of high pressure in vivo causes a hyperexcitability syndrome involving tremors and convulsions. Drugs that potentiate GABA transmission protect animals against this syndrome. It is possible that changes in γ‐aminobutyric acid (GABA) transmission may underlie the hyperexcitability. We have therefore investigated the effects of pressure on the components of GABA transmission in vitro. After incubation with [3H]GABA, hemisected frog spinal cords were superfused inside a pressure chamber and perfusate fractions were collected every 10 min. Helium, at 50 or 100 atm, did not alter the spontaneous release of GABA, but if electrical stimulation had been applied previously, then pressure caused a prolonged increase in GABA release. Helium at 50 atm did not alter the evoked release during electrical stimulation, but at 100 atm this was increased. This increase was smaller in the absence of calcium. No corresponding changes in [14C]urea efflux were seen, suggesting that the effects were not due to nonselective membrane permeability changes. The results are consistent with the known effects of pressure on neuronal activity, such as repetitive firing, but they do not suggest a selective action on the GABA release process.
Avermectin Bla Modulation of γ‐Aminobutyric Acid/Benzodiazepine Receptor Binding in Mammalian Brain
Authors: Olsen Richard W. , Snowman Adele M.
Published: October 2006

Abstract: The anthelminthic natural product avermectin Bla (AVM) modulates the binding of γ‐aminobutyric acid (GABA) and benzodiazepine (BZ) receptor ligands to membrane homogenates of mammalian brain. The potent (EC50= 40 nM) enhancement by AVM of [3H]diazepam binding to rat or bovine brain membranes resembled that of barbiturates and pyrazolopyridines in being inhibited (partially) by the convulsants picrotoxin, bicuculline, and strychnine, and by the anticonvulsants phenobarbital and chlormethiazole. The maximal effect of AVM was not increased by pentobarbital or etazolate. However, AVM affected BZ receptor subpopulations or conformational states in a manner different from pentobarbital. Further, unlike pentobarbital and etazolate, AVM did not inhibit allosterically the binding of the BZ receptor inverse agonist [3H]β‐carboline‐3‐carboxylate methyl ester, nor did it inhibit, but rather enhanced, the binding of the cage convulsant [35S]t‐butyl bicyclophosphorothionate to picrotoxin receptor sites. AVM at submicromolar concentrations had the opposite effect of pentobarbital and etazolate on GABA receptor binding, decreasing by half the high‐affinity binding of [3H]GABA and related agonist ligands, and increasing by over twofold the binding of the antagonist [3H]bicuculline methochloride, an effect that was potentiated by picrotoxin. AVM also reversed the enhancement of GABA agonists and inhibition of GABA antagonist binding by barbiturates and pyrazolopyridines. These overall effects of AVM are unique and require the presence of another separate drug receptor site on the GABA/BZ receptor complex.
Dopamine D2 Receptors in the Anterior Pituitary: A Single Population Without Reciprocal Antagonist/Agonist States
Authors: George Susan R. , Watanabe Masayuki , Seeman Philip
Published: October 2006

Abstract: Although dopamine agonists can recognize two states of the D2 dopamine receptor in the anterior pituitary (Dhigh2 and Dlow2), we examined whether the dopamine antagonists such as [3H]spiperone could recognize these two sites with different affinities. Using up to 30 concentrations of [3H]spiperone, however, we could only detect a single population of binding sites (porcine anterior pituitary homogenates) with a dissociation constant (KD) of 130 pM. When specific [3H]spiperone binding was defined by a low concentration of (+)‐butaclamol (100 nM), the apparent density was low. When defined by a high concentration of (+)‐butaclamol (10 μM), nonspecific sites became detectable, thus revealing two apparent populations of sites for [3H]spiperone, only one of which was specific for dopamine. Sodium chloride reduced the KD of the single population of specific D2 sites to 64 pM. Guanine nucleotide by itself had no effect on the KD, but enhanced the density by 25%. Since the density‐enhancement could be eliminated by extensive washing of membranes, and could be restored by preincubation with dopamine, the nucleotide‐induced elevation of D2 density appeared to be a result of the release of tightly bound endogenous dopamine. Thus, monovalent cations and guanine nucleotides appear to have separate regulatory effects on the anterior pituitary D2 receptor that modulate antagonist‐receptor interactions. Several maneuvers were used to test whether [3H]spiperone could differentiate between the two agonist‐detected subpopulations of sites. Twentyfold different concentrations of [3H]spiperone (47 pM and 1000 pM) were found to label identical proportions of receptors in the Dhigh2 and Dlow2 states as detected by the agonist 6,7‐dihydroxyaminotetralin (ADTN), suggesting that spiperone labelled equal proportions of Dhigh2 and Dlow2 sites without differential affinity for them. In addition, competition of spiperone for Dhigh2 sites selectively labelled by the agonist [3H]n‐propylnora pomorphine (NPA) had a virtually identical KD for spiperone as did the total D2 receptor population as determined by direct binding studies (75 pM versus 64 pM). [3H]Spiperone also bound to a uniform population of Dlow2 sites induced by preincubation with guanine nucleotide with identical affinity as to the total D2 population. Thus, these data do not support a “reciprocal model’ for the D2 receptor (i.e., antagonist having low affinity for Dhigh2 and high affinity for Dlow2 in a manner reciprocal to agonists). It is therefore concluded from these studies that spiperone recognizes the Dhigh2 and Dlow2 states of the receptor with equal affinity and no evidence is provided in support for reciprocal modulation of antagonist/agonist affinities.
Membrane Disordering by Anesthetic Drugs: Relationship to Synaptosomal Sodium and Calcium Fluxes
Authors: Harris R. Adron , Bruno Patrick
Published: October 2006

Abstract: The effects of membrane perturbants (ethanol, pentobarbital, chloroform, diethylether, phenytoin, cis‐vaccenic acid methylester, and cis‐vaccenoyl alcohol) on the lipid order of mouse brain synaptic plasma membranes (SPM) were tested by fluorescence polarization using 1,6‐diphenyl‐1,3,5‐hexatriene (DPH) as a probe of the membrane core and 1‐[4‐(trimethylammonium)phenyl]‐6‐phenyl‐1,3,5‐hexatriene (TMA‐DPH) as a probe of the membrane surface. The compounds decreased the fluorescence polarization of both probes, indicating that they disordered the membrane lipids. The decrease in polarization was, however, greater for DPH than for TMA‐DPH, suggesting a greater effect on the membrane core than on the membrane surface. The voltage‐dependent uptake of 24Na and 45Ca was studied in isolated mouse brain synaptosomes as a measure of membrane function. All of the compounds inhibited sodium influx, and their potencies for decreasing sodium uptake and fluorescence polarization of DPH were linearly correlated (r = 0.91). The relationship between changes in sodium influx and TMA‐DPH polarization was less consistent (r = 0.66). Synaptosomal calcium uptake was inhibited by most, but not all, of the perturbants, but this inhibition was poorly correlated with changes in fluorescence polarization of DPH (r = 0.36) or TMA‐DPH (r = 0.26). These results indicate that the function of synaptic sodium channels is correlated with lipid order in the hydrophobic core of the membrane and that the inhibitory effects of intoxicant‐anesthetic drugs on neuronal sodium fluxes may be the result of their capacity to disorder these lipids. In contrast, the effects of drugs on voltage‐dependent calcium channels were not clearly related to the capacity of these agents to disorder membrane lipids.
A New Rapid and Sensitive Bioluminescence Assay for Monoamine Oxidase Activity
Authors: Tenne M. , Finberg J. P. M. , Youdim M. B. H. , Ulitzur S.
Published: October 2006

Abstract: The in vivo luminescence of an aldehyde‐requiring mutant of the luminous bacteria Vibrio harveyi (M42) increases dramatically upon the addition of longchain aliphatic aldehydes (C8–C16). The intensity of this luminescence is linearly related to aldehyde concentration. This property was utilized for the determination of monoamine oxidase activity using n‐octylamine and n‐decylamine as substrates, which are converted by monoamine oxidase to n‐octylaldehyde and n‐decylaldehyde, respectively. The addition of the amine to a suspension containing rat liver mitochondria and M42 cells initiated a luminescence that was directly proportional to monoamine oxidase activity according to two parameters: (1) the rate of the initial increase in luminescence and (2) the final “steady‐state”level of luminescence. The new assay has advantages of high sensitivity, rapidity, the possibility to perform discontinuous as well as continuous monitoring of monoamine oxidase activity, and applicability to turbid preparations.
Is Ca2+ ‐Calmodulin‐Dependent Protein Phosphorylation in Rat Brain Modulated by Carboxylmethylation?
Authors: Billingsley Melvin L. , Velletri Paul A. , Lovenberg Walter , Kuhn Donald , Goldenring James R. , DeLorenzo Robert J.
Published: October 2006

Abstract: Calmodulin stimulation of protein kinase activity in calmodulin‐depleted preparations of rat brain cytosol or synaptosomal membranes was attenuated by prior carboxylmethylation of the enzyme source with purified protein‐O‐carboxylmethyltransferase. Similarly, calmodulin stimulation of highly purified Ca2+‐calmodulin‐dependent protein kinase was reduced if the kinase was exposed to methylating conditions prior to addition of calmodulin. Biochemical and acidic sodium dodecyl sulfate‐gel electrophoretic analyses indicated that all sources of protein kinase activity were substrates for methylation. The specific activity of methyl group incorporation into protein kinase increased with increasing purity of the preparation, reaching values of 1.72 pmol CH3/μg protein or 0.15–1.12 mol CH3/mol of holoenzyme. Analysis of ATP binding in cytosol with the use of the photoaffinity probe [32P]8‐azido‐ATP indicated that carboxylmethylation reduced ATP binding. These results suggest that carboxylmethylation of Ca2+‐calmodulin protein kinase may modulate the activity of this enzyme in rat brain.
Ascorbic Acid in Fetal Rat Brain
Authors: Kratzing C. C. , Kelly J. D. , Kratzing J. E.
Published: October 2006

Abstract: Ascorbic acid in fetal rat brain increases from 374 mg/g on the 15th day of gestation to 710 mg/g by the 20th day and remains at that level until birth. There is an 18% drop from this plateau after birth.
Characterization of a Labile Naloxone Binding Site (λ Site) in Rat Brain
Authors: Grevel Joachim , Yu Victor , Sadée Wolfgang
Published: October 2006

Abstract: A high‐affinity binding site selective for naloxone and other 4,5‐epoxymorphinans (λ site) has been previously described in rat brain. Following homogenization of freshly dissected brain, the λ sites convert from a high‐affinity to a low‐affinity state. When measured with [3H]naloxone, the decay is very rapid at 20°C (t1/2 < 2 min), whereas it is progressively slowed at lower temperatures. Proteinase inhibitors, antoxidants, and sulfhydryl group‐protecting agents failed to prevent this conversion. Kinetic measurements of μ and λ binding at varying temperatures demonstrated that the decrease in λ binding does not coincide with the concurrent increase in μ binding and that the loss of high‐affinity λ binding at 20°C can be partially restored when the temperature is lowered to 0°C. The low‐affinity state of the λ site is rather stable in the Tris buffer homogenates and is susceptible to digestion by a protease. The (–)‐isomer of WIN 44,441, a benzomorphan drug, binds to λ sites with moderate affinity (dissociation constant, KD= 63 nM), whereas the (+)‐isomer does not (KD > 10,000 nM), thus establishing stereoselectivity of the binding process. Neither the high‐affinity nor the low‐affinity state of λ binding is significantly affected by the presence of 100 mM sodium chloride or 50 μM Gpp(NH)p, (a GTP analog), which is in contrast to the dramatic effect of these agents on the established opioid receptor system. Naltrexone, naloxone, nalorphine, and morphine (in this order of decreasing potency) bind to the λ site in vivo in intact rat brain over dosage ranges that are commonly employed in pharmacological studies.
Barbiturates Are Selective Antagonists at A1 Adenosine Receptors
Authors: Lohse M. J. , Klotz K.‐N. , Jakobs K. H. , Schwabe U.
Published: October 2006

Abstract: Barbiturates in pharmacologically relevant concentrations inhibit binding of (R)‐N6‐phenylisopropyl[3H]adenosine ([3H]PIA) to solubilized A1 adenosine receptors in a concentration‐dependent, stereospecific, and competitive manner. Ki values are similar to those obtained for membrane‐bound receptors and are 31 μM for (±)‐5‐(1,3‐dimethyl)‐5‐ethylbarbituric acid [(±)‐DMBB] and 89 μM for (±)‐pentobarbital. Kinetic experiments demonstrate that barbiturates compete directly for the binding site of the receptor. The inhibition of rat striatal adenylate cyclase by unlabelled (R)‐N6‐phenyl‐isopropyladenosine [(R)‐PIA] is antagonized by barbiturates in the same concentrations that inhibit radioligand binding. The stimulation of adenylate cyclase via A2 adenosine receptors in membranes from N1E 115 neuroblastoma cells is antagonized only by 10–30 times higher concentrations of barbiturates. It is concluded that barbiturates are selective antagonists at the A1 receptor subtype. In analogy to the excitatory effects of methylxanthines it is suggested that A1 adenosine receptor antagonism may convey excitatory properties to barbiturates.
Synthesis of γ‐Glutamyldopamine and Other Peptidoamines in the Nervous System of Aplysia californica
Authors: McCaman Marilyn W. , Stetzler Judy , Clark Brian
Published: October 2006

Abstract: The synthesis of a series of γ‐glutamyl amines (γ‐Glu‐amines), including γ‐Glu‐dopamine, γ‐Glu‐5‐hydroxytryptamine, γ‐Glu‐octopamine, γ‐Glu‐tryptamine, γ‐Glu‐tyramine, and γ‐Glu‐phenylethylamine, by nervous tissue of the marine mollusc Aplysia californica is described. After ganglia were incubated in vitro with 14C‐amines, the unchanged amine and a new 14C‐labeled product, identified as the γ‐Glu conjugate of the amine, were isolated from the tissue extracts. Identification was made by comparing the chromatographic properties (HPLC, TLC, and LC) of the isolated conjugates with chemically synthesized γ‐Glu‐amines before and after acid hydrolysis.
Lipid Composition of the Nervous System of Earthworms (Lumbricus terrestris)
Authors: Okamura Nobuyuki , Stoskopf Michael , Yamaguchi Hisao , Kishimoto Yasuo
Published: October 2006

Abstract: As part of a systematic study of the evolution of the nervous system, the lipid composition of the ventral nerves of earthworms was examined. The nerve axons are wrapped in copious layers of loosely bound membrane, superficially resembling the myelin sheath of vertebrates. However, neither galactocerebroside nor sulfatide, both of which are considered to be markers for myelin, was present, and only traces of glucocerebroside, which is abundant in shrimp nerve, were detected. The lipids were rich in cholesterol (15.3 μmol/g of fresh tissue) and phospholipids (21.7 μmol/g of fresh tissue). The phospholipids were composed of phosphatidylethanolamine, ‐choline, ‐serine, and ‐inositol in the ratio of 41:44:8:7. Most of the ethanolamine‐containing phospholipids were in the form of plasmalogens. The fatty acid moieties of these phospholipids were predominantly 18:1, 18:0, and 20:1, whereas the aldehyde moieties of plasmalogen were mostly 18:0. Sphingomyelin, which is considered a ubiquitous component of animal membranes, was not detected. How the unique structure of the membranes of earthworm nerves may be related to the function of the nervous system in this organism is discussed.
N‐Methyl‐4‐Phenyl‐1,2,3,6‐Tetrahydropyridine Increases Acetylcholine and Decreases Dopamine in Mouse Striatum: Both Responses Are Blocked by Anticholinergic Drugs
Authors: Hadjiconstantinou M. , Cavalla D. , Anthoupoulou E. , Laird H. E. , Neff N. H.
Published: October 2006

Abstract: The neurotoxin N‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) produces neuropathology and clinical symptoms that resemble Parkinsonism in primates and humans. In mice it induces a long‐lasting depletion of neostriatal 3,4‐dihydroxyphenylethylamine (dopamine) content. Using the mouse, we found that MPTP induces a fall of dopamine and a rise of acetylcholine in the neostriatum. Both responses to MPTP can be blocked by prior treatment with atropine or trihexyphenidyl.
Leukotriene C4 Transport and Metabolism in the Central Nervous System
Authors: Spector Reynold , Goetzl Edward J.
Published: October 2006

Abstract: The transport and metabolism of radiolabeled leukotriene (LT) C4 in the CNS were investigated after intraventricular injection. Under thiopental (Pentothal) anesthesia, New Zealand white rabbits were injected intracerebroventricularly with 0.2 ml of artificial CSF containing 2.5 μCi of [3H]LTC4 (36 Ci/mmol), 0.3 μCi of [4C]mannitol, and, in some cases, 0.9 mg of probenecid, 1.8 mg of cysteine, 1.4 μg of unlabeled LTC4, or 2 mg of tolazoline HCl. After 2 h, the conscious rabbits were killed, and the quantity and nature of the 3H and 14C were determined in CSF, choroid plexus, and brain. The [3H]LTC4 recovered in CSF and brain was not extensively metabolized, as >70% of the 3H remained [3H]LTC4, although some spontaneous conversion to 11‐trans‐[3H]LTC4 occurred. Oxidized forms of [3H]LTC4, [3H]LTD4, and [3H]LTE4 did not exceed 18% in CSF and brain. After intraventricular injection of [3H]LTC4, 3H was transferred from the CSF to blood by a probenecidsensitive, but tolazoline‐insensitive, transport system in the CNS much more rapidly than mannitol. Cysteine decreased the retention of [3H]LTC4 in brain. These results are consistent with previous in vitro observations that [3H]LTC4 is transferred from CSF into blood by an efficient transport system for LTC4 in choroid plexus.
Differential Sensitivity of Basal and Opioid‐Stimulated Low Km GTPase to Guanine Nucleotide Analogs
Authors: Vachon Luc , Costa Tommaso , Herz Albert
Published: October 2006

Abstract In membranes derived from NG108–15 cells, the opioid peptide [D‐Ala2,D‐Leu5]enkephaIin (DADLE) stimulates a low Km GTPase. The nucleotide analogs guanosine 5′‐O‐(2‐thio)diphosphate (GDPβS), guanosine 5′‐(β,γ‐imi‐do)triphosphate [Gpp(NH)p] and guanosine 5′‐O‐(3‐thio)‐triphosphate (GTPγS) inhibit the basal enzymatic activity with the order of potency GTPγS > Gpp (NH)p > GDPβS. In the presence of DADLE, the inhibition isotherms of GDPβS and Gpp(NH)p are shifted to the right five‐ and fourfold, respectively, compared to the inhibition observed in the absence of DADLE. In contrast, the IC50 of GTPγS for inhibiting the enzyme is reduced by 55% in the presence of the opioid. Both Gpp(NH)p and GTPγS produce a concentration‐dependent increase in the Km(app) of GTPase. without affecting its Vmax, indicating a competitive inhlbi‐tion. However, the replots of Km(app) versus inhibitor concentration are hyperbolic, suggesting a partial type of inhibi‐tion. Both Gpp(NH)p and GTPγS, but not GTP, induce an increase in the EC50 of DADLE for stimulating GTPase These findings indicate that the basal and the opioid‐stimu‐lated low Km GTPase differ in their respective sensitivities to inhibition by guanine nucleotide analogs.