bluuebunny/crossref_metadata_2025 huggingface dataset:
Acetylation of Synaptosomal Protein: Inhibition by Veratridine
Authors: Berl Soil , Nunez Ramiro , Colon Arlene D. , Clarke Donald D.
Published: December 2006

Abstract: Incubation of synaptosomes with [3H]acetate results in rapid labeling of protein. Labeling is decreased in the presence of veratridine, and the effect of veratridine is blocked by tetrodotoxin. Most of the radioactivity can be removed by base or acid hydrolysis, and is probably incorporated as acetate; it is this fraction that is affected by the veratridine. The data suggest that veratridine stimulates deacetylation of synaptosomal protein. This raises the question whether acetylation‐deacetylation is involved in membrane function.
Characterization of the Solubilized GABA and Benzodiazepine Receptors from Various Regions of Bovine Brain
Authors: Asano Tomiko , Yamada Yasukazu , Ogasawara Nobuaki
Published: December 2006

Abstract: GABA and benzodiazepine receptors were solubilized from bovine cerebral cortex, cerebellum, and hippocampus and then partially purified by gel filtration and characterized. The apparent molecular weights of all these receptors were determined to be 600,000–650,000 by gel filtration, the sedimentation coefficients being 11.0–11.3 S by sucrose density gradient centrifugation. [3H]Muscimol was bound to two classes of sites in fractions from all three regions, and [3H]flunitrazepam bound to one class of sites. A comparison of the ratios of Bmax for fiunitrazepam binding to Bmax for muscimol binding revealed that the fractions from the hippocampus exhibited a much higher ratio of benzodiazepine binding sites than were detected in fractions from the cortex and cerebellum. GABA agonist and antagonist inhibited [3H]muscimol binding to the fractions from these three regions, at similar concentrations. Benzodiazepine agonists and antagonists also inhibited [3H]flunitrazepam binding in these three fractions, with similar potency. CL 218,872, however, inhibited [3H]flunitrazepam binding in the cerebellar fraction with the lowest IC50 value and that in the hippocampal fraction with the highest IC50 value. Hill coefficients for CL 218,872 inhibition were 0.98, 0.64, and 0.58 for cerebellum, cortex, and hippocampus, respectively.
Determination of Normetanephrine, 3,4‐Dihydroxyphenylethyleneglycol (Free and Total), and 3‐Methoxy‐4‐Hydroxyphenylethyleneglycol (Free and Total) in Rat Brain by High‐Performance Liquid Chromatography with Electrochemical Detection and Effects of Drugs on Regional Concentrations
Authors: Westerink Ben H. C.
Published: December 2006

Abstract: A new, fast and sensitive assay for normeta‐nephrine (NM), free and total 3,4‐dihydroxyphenylethy‐leneglycol (DOPEG), and free and total 3‐methoxy‐4‐hy‐droxyphenylethyleneglycol (MOPEG) in brain tissue is described. The method is based on high‐performance liquid Chromatography with electrochemical detection. Small Sephadex G 10 columns were used for prepurifi‐cation. This permitted the additional isolation and quantification of tyrosine, 3,4‐dihydroxyphenylalanine, nor‐adrenaline, dopamine, 3‐methoxytyramine, 3,4‐dihy‐droxyphenylacetic acid, homovanillic acid, and 5‐hydroxyindoleacetic acid. The compounds were determined in six brain areas (striatum, cortex, hippocampus, hypothalamus, brainstem, and cerebellum). Most DOPEG and MOPEG in rat brain was present in the conjugated form, except for the cerebellum, where about 80% of MOPEG was nonconjugated. No postmortem effects on MOPEG levels were observed; a slight increase in DOPEG in certain brain areas was found in microwave‐killed rats. The effects of clonidine, yohimbine, N, N‐di‐propyl‐5,6‐ADTN, and chlorpromazine on the concentrations of the five noradrenaline (NA) metabolites were determined. Free and total DOPEG and MOPEG provide similar information on NA metabolism, whereas NM (after monoamine oxidase inhibition) reflects a different type of interaction of drugs with NA metabolism. The similarity in the pattern of drug‐induced changes in NA metabolism in the various brain areas suggests that adrenoreceptors mediating NA metabolism are homogeneously distributed throughout the brain.
Regional and Subcellular Distribution in Mammalian Brain of the Enzymes Producing Adenosine
Authors: Nagata Hiroshi , Mimori Yasuyo , Nakamura Shigenobu , Kameyama Masakuni
Published: December 2006

Abstract: The activities of 5′‐nucleotidase, 2′‐nucleotid‐ase, alkaline phosphatase, and acid phosphatase were measured in rat and autopsied human brains. The four phosphatases in the rat brain showed little change in activity after death. The activities of adenosine‐producing enzymes were compared in various parts of rat and human brains. When phosphatase activity was measured at pH 7.5, 5′‐nucleotidase showed the highest activity in the most parts of the brain. The activity of 2′‐nucleotidase and that of nonspecific phosphatase were almost the same at pH 7.5. However, higher phosphatase activity was observed in all parts of the brain when nonspecific phosphatase activity was measured at pH 10.0 or 5.5. High specific activity of 5′‐nucleotidase in the brain was detected in the membranous components, especially in the synaptic membranes. The activity of 2′‐nucleotidase was distributed in the soluble and synaptosomal fractions. The highest activity of both alkaline and acid phosphatases was recovered in the crude mitochondrial fraction, with the highest specific activity in the microsomal fraction. Phosphatase activity was distributed widely in the rat brain. The activity of 5′‐nucleotidase was high in the medulla oblongata, thalamus, and hippocampus, but low in the peripheral nerve, spinal cord, and occipital lobe. The activity of 2′‐nucleotidase was high in the vermis and frontal lobe. The highest acid and alkaline phosphatase activities were detected in the frontal lobe and in the olfactory bulb, respectively. The distribution of the four phosphatases in the autopsied human brain was similar to that in the rat brain. The highest 5′‐nucleotidase activity was observed in the temporal lobe and thalamus. High activity of 2′‐nucleotidase was found in the basal ganglia, substantia nigra, and cortical motor area. Alkaline phosphatase was enriched in the temporal cortex, parietal cortex, and amygdala, and low in the white matter. High acid phosphatase activity was detected in the insula and cerebellar cortex.
Isolation and Immunological Characterization of a Glycoprotein from Adrenal Chromaffin Granules
Authors: Fischer‐Colbrie R. , Zangerle R. , Frischenschlager I. , Weber A. , Winkler H.
Published: December 2006

Abstract: A glycoprotein (s‐GP III) was isolated from the soluble lysate of chromaffin granules by chromatography with immunoaffinity and lectin columns. An identical protein (m‐GP III) was shown to be present in the granule membranes. The apparent molecular weight of these glycoproteins as determined by the electrophoresis system of Laemmli (1970) was 43,000 under reducing conditions. In the absence of mercaptoethanol they aggregated to dimers. Antisera were raised against both the soluble and the membrane‐bound forms of this glycoprotein. With these antisera GP III was further characterized: Immunoreplicas were obtained after two‐dimensional electrophoresis of soluble and membrane‐bound proteins of chromaffin granules. GP III was identified as a protein with a rather broad pI (4.6‐5.3), indicating microhetero‐geneity. As shown by subcellular fractionation, m‐GP HI is specifically confined to chromaffin granules. GP III can therefore be used as a marker for the membranes of these organelles. The soluble form is secreted from adrenal medulla during stimulation with carbamylcholine chloride. An immunologically identical antigen was detected in adeno‐ and neurohypophysis. The physiological function of GP III is still unknown. It does not demonstrate any of the enzymatic activities so far known to occur in chromaffin granules.
Isolation of Membrane Attack Complex of Complement from Myelin Membranes Treated with Serum Complement
Authors: Silverman Barry A. , Carney David F. , Johnston Curtis A. , Vanguri Padmavathy , Shin Moon L.
Published: December 2006

Abstract: The interaction between complement and myelin membranes and its possible role in myelin damage and in the disposal of damaged myelin in vivo is of interest because activation of complement generates both opsonin(s) and membrane attack complex of complement. In our studies on the role of complement in demyelin‐ation, we have shown that isolated myelin activates serum complement in the absence of myelin‐specific antibody and that membrane attack complex of complement is the required factor in antibody‐mediated demyelination of mouse cerebellar expiant cultures. In the present study, we examined whether activation of serum complement by myelin is associated with the formation of membrane attack complex of complement in myelin membranes. Extracts of myelin‐associated proteins following incubation of myelin with fresh serum were studied by ultracentrifugation on a sucrose density gradient for detection of C5b‐9 neoantigen. The subunit structure of C5b‐9 was determined by sodium dodecyl sulfate‐poly‐acrylamide gel electrophoresis, electroblotting, and immunostaining. Results indicate that the macromolecular complex consisting of late‐acting complement components, C5‐C9, was assembled in the target myelin membranes.
Cytoskeletal Structures and Oligodendroglial Differentiation in C‐6 Glial Cells
Authors: Antonow Juli A. , Bhat Narayan , Volpe Joseph J.
Published: December 2006

Abstract: The relationship of the cytoskeleton to a biochemical expression of oligodendroglial differentiation was studied in cultured C‐6 glial cells. Specifically, we investigated the effect of the cytoskeletal perturbants, colchicine and cytochalasin D, on the induction of the oligodendroglial marker enzyme. 2′,3′‐cyclic nucleotide 3′‐phosphohydrolase (CNP), caused by removal of serum from the culture medium. Each drug inhibited CNP induction in a concentration‐dependent manner, and essentially complete inhibition of induction was observed with 0.25 μM colchicine or 2.0μM cytochalasin D. Detailed study of the effect of colchicine was carried out. This antimicrotubular agent not only totally prevented induction if added at the onset of serum removal, but also prevented further induction when added at various times after serum removal. That the effect of colchicine related to the drug's effect on microtubules was supported by the demonstration that lumicolchicine, a colchicine isomer which has no effect on microtubules, had no effect on the CNP induction. Moreover, colchicine, but not lumicolchicine, prevented the morphological signs of differentiation provoked by serum removal. The effect of colchicine was reversible and relatively specific. Thus, no concomitant effect of colchicine on the activity of another plasma membrane enzyme of C‐6 cells, i.e., (Na++ K +)‐acti‐vated ATPase, or on the rate of incorporation of [3H]leucine into total protein of intact cells could be discerned. The possibility that the site of the effect of colchicine is on intracellular events was suggested by the observation that the drug inhibited the induction of CNP by dibutyryl cyclic AMP. The data suggest that the cytoskeleton is involved in oligodendroglial differentiation.
Identification of the Major Postsynaptic Density Protein as Homologous with the Major Calmodulin‐Binding Subunit of a Calmodulin‐Dependent Protein Kinase
Authors: Goldenring James R. , McGuire Joseph S. , DeLorenzo Robert J.
Published: December 2006

Abstract: The major postsynaptic density protein (mPSDp), comprising >50% of postsynaptic density (PSD) protein, is an endogenous substrate for calmodulin‐dependent phosphorylation as well as a calmodulin‐binding protein in PSD preparations. The results in this investigation indicate that mPSDp is highly homologous with the major calmodulin‐binding subunit (p) of tubulin‐associated calmodulin‐dependent kinase (TACK), and that PSD fractions also contain a protein homologous with the o‐subunit of TACK. Homologies between mPSDp and a 63,000 dalton PSD protein and the p‐ and ó‐subunits of TACK were established by the following criteria: (1) identical apparent molecular weights: (2) identical calmodulin‐binding properties; (3) manifestation of Ca2+ ‐calmodulin‐stimulated autophosphorylation; (4)identical isoelectric points; (5) identical calmodulin binding and autophosphorylation patterns on two‐dimensional gels; (6) homologous two‐dimensional tryptic peptide maps; and (7) similar phosphoamino acid‐specific phosphorylation of tubulin. The results suggest that mPSDp is a calmodulin‐binding protein involved in modulating protein kinase activity in the postsynaptic density and that a tubulin kinase system homologous with TACK exists in a membrane‐bound form in the PSD.
Effect of Denervation on a Cholinergic‐Specific Ganglioside Antigen (Chol‐1) Present in Torpedo Electromotor Presynaptic Plasma Membranes
Authors: Ferretti P. , Borroni E.
Published: December 2006

Abstract: The presence of Chol‐1, an antigen identified in the plasma membrane of cholinergic electromotor nerve terminals of Torpedo marmorata, was investigated in Torpedo electric organ after 3, 6, and 9 weeks’denervation. Denervation was monitored by the cessation of stimulus‐evoked discharge potentials, by the reduction in nerve terminals seen morphologically, and by the decrease in ACh and ChAT contents. The content of ganglioside‐bound sialic acid did not show any appreciable change with time. Some modification of ganglioside pattern on TLC was observed after 9 weeks’denervation. The presence of Chol‐1 after denervation was assayed by its activity in inhibiting the selective complement‐induced lysis of the cholinergic subpopulation of guinea pig cortical synaptosome which is mediated by the anti‐Chol‐1 antiserum. Denervation did not affect Chol‐1 immunoreactivity although it did alter the distribution of the immunoreactivity among gangliosides. The possible significance of the results is discussed.
Desipramine Elicits the Expression of Opiate Receptors and Sulfogalactosylceramide Synthesis in Rat C6 Glioma Cells
Authors: Tocque Bruno , Albouz Samia , Boutry Jeanne‐Marie , Saux Francoise Le , Hauw Jean‐Jacques , Bourdon Raymond , Baumann Nicole , Zalc Bernard
Published: December 2006

Abstract: In the course of our studies on lipidoses induced by amphiphilic drugs, we have investigated the efof desipramine, a tricyclic antidepressant, on glial cells in culture. We noted that the addition of desipramine to the culture medium of C6 glioma cells resulted in the modification of the lipid profile of the cell membranes. Of particular interest was the presence, in the desipraminetreated cells, of an additional lipid comigrating on thin layer chromatography with Sulfogalactosylceramide (S‐GalCer). Addition of radiolabelled sulfuric acid in the culture medium of the desipramine‐treated cells resulted in the incorporation of [35S]sulfate in the newly synthesized lipid. Furthermore, this lipid was localized selectively by indirect immunofluorescence using a specific rabbit anti‐S‐GalCer antibody on the cell surface of desipraminetreated. but not control, C6 cells. Desipramine also increased the activity of 3′‐phosphoadenosine‐5′‐phosphosulfate sulfotransferase (the enzyme responsible for the synthesis of S‐GalCer). Since it has been suggested that S‐GalCer may be involved in opiate receptors, we looked for opiate binding sites on C6 glioma cells after exposure to desipramine. We found that dihydromorphine was able to bind to the desipramine‐treated C6 cell membrane. The binding of [3H]dihydromorphine (180 fmol/mg protein) was stereospecific and had a KD of 30‐60 nM. Furthermore, morphine reduced both the basal and isoproterenol‐stimulated cyclic AMP levels of the desipraminetreated C6 cells. This effect was blocked by naloxone. In these respects, the opiate binding sites induced after treatment of C6 glioma cells with desipramine fulfill the requirements of a true opiate receptor.
Effect of Oxygen on the Induction of Tryptophan Hydroxylase by Adrenergic Agents in Organ Cultures of Rat Pineal Glands
Authors: Sitaram B. R. , Lees G. J.
Published: December 2006

Abstract: Rat pineal glands, cultured under 95% air and 5% CO2, lost 50% of their tryptophan hydroxylase activity within 5 h. This loss was accelerated by the addition of cycloheximide or puromycin to the medium. Activity was, however, largely maintained in 95% O2 and 5% CO2. Under these conditions, L‐noradrenaline (100 μ.M), L‐iso‐proterenol (10 μM), and dibutyryl cyclic AMP (1 mM) induced enzyme activity. They failed to do so when 95% air and 5% CO2 was used. Noradrenaline induced serotonin N‐acetyltransferase activity with either atmosphere.
Novel 1,4‐Dihydropyridine (Bay K 8644) Facilitates Calcium‐Dependent [3H]Noradrenaline Release from PC 12 Cells
Authors: Albus Udo , Habermann Ernst , Ferry David R. , Glossmann Hartmut
Published: December 2006

Abstract: The effects of the novel 1,4‐dihydropyridine Bay K 8644 [methyl‐1,4‐dihydro‐2,6‐dimethyl‐3‐nitro‐4‐(2‐trifluoromethylphenyl)‐pyridine‐5‐carboxylate] on the release of [3H]noradrenaline in cultured PC 12 cells were investigated. K+ in a concentration‐dependent manner evoked 3H‐transmitter release with an EC50 of 50‐56 mM. Bay K 8644 at 30 nM potentiated the K+‐evoked [3H]noradrenaline release; however, in the absence of calcium neither K+ evoked nor Bay K 8644 enhanced [3H]noradrenaline release. At a K+ concentration of 25 mM, Bay K 8644 stimulated [3H]noradrenaline release fivefold, with an EC50 of 10 nM, and 100 nM of the calcium channel blocker nitrendipine shifted the concentration response curve of Bay K 8644 to the right in an apparently competitive fashion. Nitrendipine blocked the Bay K 8644‐potentiated release with an EC50 of 700 nM in the presence of 500 nM Bay K 8644. [3H]Nitrendipine bound to a saturable population of binding sites on PC 12 cell membranes with a Bmax of 180 fmol μmg‐1 of membrane protein and a KD of 0.9 nM. Bay K 8644 inhibited [3H]nitrendipine binding with a K1 of 16 nM. It is concluded that Bay K 8644 binds to, and stabilizes, the open state of calcium channels and thus acts as a “calcium agonist” to mediate calcium‐dependent cellular events such as catecholamine release from PC 12 cells.
Evidence for the Neurotrophic Regulation of Collagen‐Tailed Acetylcholinesterase in Immature Skeletal Muscle by β‐Endorphin
Authors: Haynes Laurence W. , Smith Margaret E. , Smyth Derek G.
Published: December 2006

Abstract: Acetylcholinesterase (AChE) was extracted in a high‐saline medium from gastrocnemius muscles of rat embryos and young rats aged 14 days’gestation to 40 days post partum. The molecular forms of the enzyme were separated by low‐salt precipitation, followed by velocity sedimentation. During gestation, all molecular forms increased in activity, particularly the 16 S (A12) form. During the first 2 weeks of life, there was a large increase in the activity of soluble AChE (G forms), whilst the activity of insoluble AChE (A forms) was reduced. Denervation of the muscle reversed the change in the relative proportions of the molecular forms. The embryonic pattern of activities of AChE forms persisted in cultures of myotubes obtained at 20 days’gestation and maintained in the absence of spinal cord. When myotubes were maintained in medium previously conditioned by developing spinal cord explants, 16 S AChE declined while the soluble (4 and 6 S) forms increased in activity in a manner resembling that seen in early postnatal muscles in vivo. β‐Endorphin (β‐EP) immunoreactivity was detected in the spinal cord‐conditioned medium and was identified by HPLC and ion‐exchange chromatography as β‐EP‐(l–31) plus its shortened and N‐acetylated forms. Cultivation of myotubes in the presence of synthetic camel β‐EP resulted in a reversible change in the pattern of AChE forms which was similar to that seen with spinal cord‐conditioned medium. These studies provide evidence for the neuroregulation of AChE A and G forms in immature skeletal muscle. A major candidate for this role is β‐EP, produced and released by developing spinal cord.
Chromaffin Granule Membrane‐F‐Actin Interactions and Spectrin‐Like Protein of Subcellular Organelles: A Possible Relationship
Authors: Aunis Dominique , Perrin Dominique
Published: December 2006

Abstract: The membrane of chromaffin granule, the secretory vesicle of adrenal medullary cells storing catecholamines, enkephalins, and many other components, interacts with F‐actin. Using low shear falling ball viscometry to estimate actin binding to membranes, we demonstrated that mitochondrial and plasma membranes from chromaffin cells also provoked large increases in viscosity of F‐actin solutions. Mitochondrial membranes also had the capacity to cause complete gelation of F‐actin. In addition, vasopressin‐containing granules from neurohypophysial tissue were shown to bind F‐actin and to increase the viscosity of F‐actin solutions. Using an antibody directed against human erythrocyte spectrin, it was found that a spectrin‐like protein was associated with secretory granule membrane, mitochondrial membrane, and plasma membrane. The chromaffin granule membrane‐associated spectrin‐like protein faces the cytoplasmic side, is composed of two subunits (240 kD and 235kD), the α‐subunit (240 kD, pHi 5.5) being recognized by the antibody. Nonionic detergents such as Triton X‐100 or Nonidet P40 failed to release fully active spectrin‐like protein. In contrast, Kyro EOB, a different nonionic detergent, was found to release spectrin‐like protein while keeping intact F‐actin binding capacity, at least below 0.5% Kyro EOB concentration. Chromaffin cells in culture were stained with antispectrin antibody, showing the presence of spectrin‐like protein in the cell periphery close to the cell membrane but also in the cytoplasm. We conclude that in living cells the interaction of F‐actin with chromaffin granule membrane spectrin observed in vitro is important in controlling the potential function of secretory vesicles.
Synthesis of Catecholamines in the Developing Spinal Cord of the Rat
Authors: Commissiong John W.
Published: December 2006

Abstract: Using mass fragmentographic techniques, the synthesis of dopamine (DA) and norepinephrine (NE) was studied in five functional regions in the spinal cord of the developing rat. Norepinephrine was first detectable in the whole cord on fetal day (FD) 16. Thereafter, there was a rapid increase in synthesis. The fastest rate occurred in the ventral horn. The peak concentration was recorded in the latter half of neonatal life, followed by a decline back to the levels found in the adult cord. The capacity of the developing cord to synthesize NE from injected L‐dihydroxyphenylalanine (l‐DOPA) increased in concert with its normal synthetic capacity. There was no clear consistent pattern in the development of DA synthesis, with two exceptions: (1) in the cervical dorsal horn (CDH), cervical ventral horn (CVH), and thoracic zona intermedia (TZI), there was a peak of DA on the day of birth; (2) in all regions there was a peak of DA on neonatal day (ND) 20. At FD 16 (the earliest time studied) the developing cord was capable of considerable synthesis (6.0 nmol/g) of DA from injected L‐DOPA. This synthetic capacity developed rapidly, peaked at ND 4, and declined back to the adult levels by ND 20. Control experiments indicate that only about 10% (at best) of the DA synthesized from injected l‐DOPA occurs in monoaminergic nerve terminals. Norepinephrine is synthesized exclusively in noradrenergic nerve terminals. The newly synthesized DA and NE are both extensively metabolized in the cord. The results are discussed in relationship to current attempts to understand the functional importance of spinal monoaminergic nerves. Comparisons are also made with the development of monoaminergic nerves elsewhere in the central nervous system.
Antibodies to Dopamine: Radioimmunological Study of Specificity in Relation to Immunocytochemistry
Authors: Geffard M. , Kah O. , Onteniente B. , Seguela P. , Le Moal M. , Delaage M.
Published: December 2006

Abstract: Two classes of anti‐3,4‐dihydroxyphenylethylamine (dopamine) antibodies were raised in rabbits using dopamine conjugated to albumin either via formaldehyde or via glutaraldehyde. Each was usable for immunohistochemical detection of dopamine neurons provided that the tissue was fixed by the homologous cross‐linking agent. However, anti‐dopamine‐glutaraldehyde antibodies turned out to be of more general use because of the better fixative properties of glutaraldehyde which fixed dopamine in rat and in teleost, whereas formaldehyde only worked in lower vertebrates (such as goldfish) and not in rat brain. The specificity of antidopamine‐glutaraldehyde antibodies was firmly established by competition experiments in equilibrium dialysis, using an immunoreactive tritiated derivative synthesized by coupling dopamine to N‐α‐acetyl‐L‐lysine N‐methylamide via glutaraldehyde. Specificity studies in vitro and immunohistological results demonstrating the specific staining of dopaminergic neurons were found to correlate well.
Angiotensin‐Converting Enzyme Activity Is Reduced in Brain Microvessels of Spontaneously Hypertensive Rats
Authors: Kobayashi Hideyuki , Take Keiko , Wada Akihiko , Izumi Futoshi , Magnoni Maria Sandra
Published: December 2006

Abstract: Angiotensin‐converting enzyme (ACE) activity in brain microvessels of spontaneously hypertensive rats (SHR) and Wistar Kyoto (WKY) controls was measured. Cerebral microvessels, prepared from the cerebral cortices by the albumin flotation and glass bead filtration technique, were free of neuronal and glial elements. ACE activity in brain microvessels of SHR was lower than that of WKY. A Woolf‐Augustinsson‐Hofstee plot showed that the reduction of the enzyme activity in SHR was due to a 30% decrease in Vmax without any change in Km for substrate. The decrease of ACE activity in brain micro‐vessels of SHR may indicate an impairment of the central renin‐angiotensin system and may be related to cerebral microvascular dysfunctions occurring in hypertension.
Characterization, Localization, and Biosynthesis of an Interstitial Retinol‐Binding Glycoprotein in the Human Eye
Authors: Fong S.‐L. , Liou G. I. , Landers R. A. , Alvarez R. A. , Gonzalez‐Fernandez F. , Glazebrook P. A. , Lam D. M. K. , Bridges C. D. B.
Published: December 2006

Abstract: Human eyes contain an Mr 135K retinol‐binding protein that is analogous to interstitial retinol‐binding protein (IRBP) in the subretinal space of bovine eyes. It is a glycoprotein, because it binds 125I‐concanavalin A, 125I‐wheat germ agglutinin and 125I‐Lens culinaris hemagglutinin. It does not bind Ricinus communis agglutinin I. After desialation, it binds Ricinus communis agglutinin I, but loses its capacity to bind wheat germ agglutinin. These observations, coupled with the known specificities of these lectins, suggest that at least one of the oligosaccharide chains is a sialated, biantennary complex type containing fucose. Both by direct analysis of dissected ocular tissues and by immunocytochemistry it was shown that human interstitial retinol binding protein is an extracellular protein that is confined predominantly to the subretinal space. Monkey retinas incubated in vitro in medium containing [3H]leucine were shown to synthesize and secrete this protein into the medium, a conclusion that was confirmed by immunoprecipitation with an immunoglobulin fraction prepared from rabbit antibovine IRBP serum. Virtually no other labeled proteins were detectable in the medium. It is concluded that interstitial retinol‐binding protein meets many of the requirements for a putative transport protein implicated in the transfer of retinol between the pigment epithelium and retina during the visual cycle, and that the neural retina may play an important role in regulating its amount in the subretinal space.
Activation of Adenylyl Cyclase by Preincubation of Rat Cerebral‐Cortical Membranes Under Phosphorylating Conditions: Role of ATP, GTP, and Divalent Cations
Authors: Whittemore Scott R. , Graber Stephen G. , Lenox Robert H. , Hendley Edith D. , Ehrlich Yigal H.
Published: December 2006

Abstract: The effects of preincubation under phosphorylating conditions on adenylyl cyclase activity were studied in preparations containing synaptic membranes from rat cerebral cortex. Preincubation of the membranes with 2 mM ATP and 10 mM MgCl2 resulted in a 50% increase of adenylyl cyclase activity which withstood sedimentation and washing. This activation was maximal after 5 min of preincubation, was reversed after longer preincubations, and paralleled the time course of endogenous phosphorylation‐dephosphorylation of proteins observed under these conditions. The activation showed a critical requirement for Mg2+ ions and was dependent on ATP concentration. Similar activation was observed after preincubation of cerebral‐cortical membranes with adenosine‐5′‐0‐(3‐thiophosphate) (ATPγS), but this activation was not reversed by prolonged preincubation times. The activation by ATPγS was potentiated severalfold by including synaptoplasm in the preincubation. Further experiments indicated that the activity of nucleoside diphosphokinase, which converts ATPγS to guanosine‐5′‐0‐(3‐thiophosphate) (GTPγS), could account for this potentiation. Preincubation of washed membranes for 5 min with 10 μ.M GTP and 10 mM MgCl2 also produced a 50% activation of adenylyl cyclase which withstood sedimentation and washing and was reversed by longer preincubations. Endogenous phosphorylation of specific protein components in the membranes during the preincubation was examined by including radioactively labeled nucleoside thiophosphates in the preincubation medium. Incorporation of 35S from [35S]ATPγS into a protein component with apparent Mr of 54,000 daltons (54K) correlated significantly with the activation of adenylyl cyclase by ATPγS. Thiophosphorylation of the 54K protein was potentiated by addition of GDP to reactions carried out with [35S]ATPγS. Endogenous activity utilizing [γ‐32P]GTP as a phosphate donor also preferentially phosphorylated the 54K protein band. These results support previous suggestions that protein phosphorylation plays a role in the regulation of adenylyl cyclase activity. Among the numerous membrane‐bound phosphoproteins in rat brain, we have identified a specific protein component with an apparent Mr of 54,000 daltons as the most likely candidate for involvement in this mode of regulation. This 54K protein, which is a principal substrate for a GTP‐preferring protein kinase activity in brain membranes, can now be at the focus of investigations attempting to demonstrate a direct role for protein phosphorylation in adenylyl cyclase regulation.
Characterization of γ‐Aminobutyric Acid‐Benzodiazepine Receptor Complexes by Protection Against Inactivation by Group‐Specific Reagents
Authors: Maksay Gabor , Ticku Maharaj K.
Published: December 2006

Abstract: The chemical topography of the γ‐aminobutyric acid (GABA) and benzodiazepine (BZ) receptors was investigated in a thoroughly washed cortical membrane preparation of the rat. Chemical modification by several amino‐ and tyrosyl‐selective reagents and the protection from it by direct and allosteric ligands of the GABA‐BZ receptor complex were used to identify the residues at the binding sites. Inhibition of specific GABA binding by p‐diazobenzenesulfonic acid (DSA), tetrani‐tromethane (TNM), and N‐acetylimidazole and the selective and complete protection from it by GABA and muscimol suggest the presence of a tyrosine residue at the GABAA site. TNM, like DSA, selectively decreased the number of the low‐affinity GABA receptors, and this could be completely protected only by GABA concentrations that can saturate the low‐affinity sites. TNM pre‐treatment also abolished the muscimol enhancement of [3H]diazepam binding, which suggests that the low‐affinity GABA receptor sites are responsible for this enhancement. Inhibition of GABA binding by pyridoxal‐5‐phosphate (PLP) and the selective protection by GABA and muscimol support the presence of a lysine residue at the GABAA receptor site. Complete and selective protection from diethylpyrocarbonate (DEP) inhibition of [3H]diazepam binding by flurazepam suggests the presence of a histidine residue at the BZ site. Flurazepam selectively protected from inhibition of [3H]diazepam binding by N‐bromosuccinimide and N‐acetylimidazole, but not that by DSA and TNM, which does not allow a unanimous conclusion regarding the presence of tyrosine or tryptophan residues at the BZ site.
Opiates Inhibit Acetylcholine Release from Torpedo Nerve Terminals by Blocking Ca2+ Influx
Authors: Michaelson D. M. , McDowall G. , Sarne Y.
Published: December 2006

Abstract: In the present communication we report that Ca2+‐dependent acetylcholine release from K+‐depolarized Torpedo electric organ synaptosomes is inhibited by morphine, and that this effect is blocked by the opiate antagonist naloxone. This finding suggests that the purely cholinergic Torpedo electric organ neurons contain pre‐synaptic opiate receptors whose activation inhibits acetylcholine release. The mechanisms underlying this opiate inhibition were investigated by comparing the effects of morphine on acetylcholine release induced by K+ depolarization and by the Ca2+ ionophore A23187 and by examining the effect of morphine on 45Ca2+ influx into Torpedo nerve terminals. These experiments revealed that morphine inhibits 45Ca2+ influx into K+‐depolarized Torpedo synaptosomes and that this effect is blocked by naloxone. The effects of morphine on K+ depolarization‐mediated 45Ca2+ influx and on acetylcholine release have similar dose dependencies (half‐maximal inhibition at 0.5–1 μM), suggesting that opiate inhibition of release is due to blockage of the presynaptic voltage‐dependent Ca2+ channel. This conclusion is supported by the finding that morphine does not inhibit acetylcholine release when the Ca2+ channel is bypassed by introducing Ca2+ into the Torpedo nerve terminals via the Ca2+ ionophore.
Depolarisation‐Dependent Protein Phosphorylation in Rat Cortical Synaptosomes Is Inhibited by Fluphenazine at a Step After Calcium Entry
Authors: Robinson Phillip J. , Jarvie Paula E. , Dunkley Peter R.
Published: December 2006

Abstract: The sequence of molecular events linking depolarisation‐dependent calcium influx to calcium‐stimulated protein phosphorylation is unknown. In this study the effect of the neuroleptic drug fluphenazine on depolarisation‐dependent protein phosphorylation was investigated using an intact postmitochondrial pellet isolated from rat cerebral cortex. Fluphenazine, in a dose‐dependent manner, completely inhibited the increases in protein phosphorylation observed previously. The concentration of fluphenazine required for 50% inhibition varied for different phosphoproteins but for synapsin I was 123 μM. Other neuroleptics produced effects similar to fluphenazine with their order of potency being thioridazine > haloperidol > trifluoperazine > fluphenazine > chlorpromazine. Fluphenazine also increased the phosphorylation of proteins in nondepolarised controls at concentrations of 20 and 60 μM. The inhibition of depolarisation‐dependent phosphorylation was apparently not due to a loss of synaptosomal integrity or viability, a decrease in calcium uptake, a change in substrate availability, or to a change in protein phosphatase activity. The data are most consistent with an inhibition of protein kinase activity by blockade of calmodulin or phospholipid activation.
Portacaval Anastomosis: Brain and Plasma Metabolite Abnormalities and the Effect of Nutritional Therapy
Authors: Mans Anke M. , Biebuyck Julien F. , Davis Donald W. , Hawkins Richard A.
Published: December 2006

Abstract: Rats with portacaval shunts were used as a model of hepatic encephalopathy and compared to sham‐operated controls. First, the changes in intermediary metabolites and amino acids in blood and whole brain were characterized and found to be similar at 4 and 7 weeks after shunting. Second, the effects of nutritional therapy on selected metabolites and tryptophan transport into brain were assessed in rats 5 weeks after surgery. Ordinary food was removed and the rats were treated with glucose given either by mouth or intravenously, or intravenous glucose plus branched chain amino acids. Several abnormalities in plasma amino acid concentrations were reversed by treatment. The abnormally high brain uptake index of tryptophan, a consequence of portacaval shunting, was not lowered by any of the treatment regimens; it was even higher in the groups given glucose by mouth and glucose plus amino acids. Calculated competition for entry of tryptophan, phenylalanine, and tyrosine into brain was unchanged (glucose plus amino aicds), or reduced (glucose alone). Brain glutamine content was brought to near normal by all treatments. Infusion of glucose plus branched chain amino acids normalized brain content of tryptophan, phenylalanine, and tyrosine, even though the brain uptake index of tryptophan was higher in this group. Thus, partial or complete reversal of several abnormalities found after portacaval shunting was achieved by removal of oral food and administration of glucose. The addition of branched chain amino acids to the glucose infusion restored brain content of three aromatic amino acids to near normal, by a mechanism which appeared to be unrelated to transport across the blood‐brain barrier.
Human Brain Phenol Sulfotransferase: Biochemical Properties and Regional Localization
Authors: Young William F. , Okazaki Haruo , Laws Edward R. , Weinshilboum Richard M.
Published: December 2006

Abstract: Phenol sulfotransferase (PST) catalyzes the sulfate conjugation of catecholamines and phenol and catechol drugs. The human blood platelet contains a thermolabile (TL) form of PST that catalyzes the sulfate conjugation of dopamine and other monoamines and a thermostable (TS) form that catalyzes the sulfate conjugation of micromolar concentrations of phenol and p‐nitrophenol. Experiments were performed to determine whether the brain contains forms of PST analogous to the TL and TS forms found in the human platelet, and to determine whether there are regional variations in human brain PST activity. We found that the human brain contains at least two forms of PST, forms that are similar to the platelet TS and TL forms of the enzyme with respect to substrate specificity, apparent Km constants, thermal stability, and sensitivity to inhibitors. Optimal conditions were determined for the measurement of these two activities in brain homogenates. The stability of PST activities in the human brain after death was determined in five samples of cerebral cortex that were obtained during clinically indicated neurosurgical procedures. An average of 76 ± 8% and 80 ± 9% (mean ± SEM) of the basal TL and TS PST activities, respectively, remained in these five samples of cerebral cortex after 8 h of storage under simulated post‐mortem conditions. Six human brains were then obtained less that 8 h after death from patients who had no neurological disease prior to death. The mean activities of the TL and TS forms of PST were measured in 17 different regions of the six brains. If the pituitary was excluded from consideration, TL and TS PST activities both varied approximately fivefold among these regions, and both activities were highest in cerebral cortex. However, the average TS activity in the anterior pituitary, a tissue of non‐neural origin embryologically, was 6.5‐fold greater than the highest average TS PST activity found in cerebral cortex.
High Doses of Vitamin B6 in the Rat Are Associated with Inhibition of Hepatic Tryptophan Metabolism and Increased Uptake of Tryptophan into the Brain
Authors: Bender David A. , Totoe Lena
Published: December 2006

Abstract: Acute administration of vitamin B6 to rats (10 mg/kg body weight) led to reduced urinary excretion of N1‐methyl nicotinamide and methyl pyridone carboxamide, indicating inhibition of the oxidative metabolism of tryptophan. There was a considerable reduction in the production of 14CO2 from [ring‐2‐14C]tryptophan, and a significant inhibition of hepatic tryptophan oxygenase when measured in liver homogenates, together with an increase in the concentration of tryptophan in plasma. There was an increase in both the concentration of tryptophan in the brain and the uptake into the brain of peripherally administered [3H]tryptophan, accompanied by a small increase in the rate of synthesis of 5‐hydroxy‐tryptamine in the brain. It is suggested that this increase in the uptake of tryptophan into the brain following a relatively large dose of vitamin B6 may explain the beneficial action of the vitamin in some cases of depressive illness.
Modulation of Receptors for Thyrotropin‐Releasing Hormone by Benzodiazepines: Brain Regional Differences
Authors: Sharif Najam A. , Burt David R.
Published: December 2006

Abstract: The benzodiazepines (BZDs) chlordiazepoxide (CDE), diazepam (DZM), and flurazepam (FLM) inhibited receptor binding for thyrotropin‐releasing hormone (TRH) with low micromolar potency. In contrast, numerous other categories of drugs were previously shown to be inactive. Scatchard analysis of competition data suggested that the BZDs reduced TRH receptor affinity, consistent with competitive inhibition. Receptors from amygdala, retina, and pituitary appeared more sensitive to inhibition by BZDs than those from hypothalamus, hippocampus, spinal cord, or cerebellum. The latter four regions also gave shallower inhibition curves. CDE revealed an apparently biphasic dissociation of [3H][3‐Me‐His2]TRH([3H]MeTRH) from amygdala membranes at 4°C, with kinetics similar to those with TRH. These results suggest that TRH receptors in the brain are heterogeneous and that certain BZDs in high therapeutic concentrations may exert central effects through actions at TRH receptors or coupled proteins.
An Evaluation of Irreversible Inhibition of Synaptosomal High‐Affinity Choline Transport by Choline Mustard Aziridinium Ion
Authors: Rylett R. Jane , Colhoun E. Howard
Published: December 2006

Abstract: Choline mustard aziridinium is a potent, irreversible and selective blocker of sodium‐dependent, high‐affinity transport of choline into rat forebrain synaptosomes; it was found to be 30 times less potent against low‐affinity transport of choline. The IC50 value for high‐affinity transport was 0.94 μM, compared to 29 μM for low‐affinity uptake. The inhibitory action of choline mustard aziridinium ion on high‐affinity transport of choline was graded with respect to time; a 12‐fold increase in potency was obtained by increasing the inhibitor preincubation times from 1 to 30 min. Low concentrations of choline mustard aziridinium ion could produce significant blockade of choline carriers providing the exposure time was prolonged. The characteristics of the blockade of synaptosomal high‐affinity choline transport by choline mustard aziridinium ion also changed depending upon preincubation time. The kinetics of inhibition of high‐affinity choline transport by choline mustard aziridinium ion showed apparent competitive inhibition initially, followed by noncompetitive characteristics at longer preincubations with inhibitor. The rate of irreversible inhibition of carriers by this nitrogen mustard analogue would appear to be rapid; the rate constant was determined to be 5 × 10−2 s−1for micromolar concentrations of inhibitor. This action may preclude the transport of the mustard analogue into the nerve terminal, although initially some reversible binding with the carrier may result in the translocation of some choline mustard aziridinium ion into the presynaptic ending. The progressive alkylation of high‐affinity carriers by the analogue could indicate the presence of excess carrier sites in the presynaptic membrane, or subpopulations of carriers in an inactive state in equilibrium with active carriers. A model is described for the inhibitory action of choline mustard aziridinium ion on synaptosomal high‐affinity choline carriers.
Cerebral Vascular Adenylate Cyclase: Evidence for Coupling to Receptors for Vasoactive Intestinal Peptide and Parathyroid Hormone
Authors: Huang Minta , Rorstad O. P.
Published: December 2006

Abstract: We have studied the responsiveness of vascular adenylate cyclase to vasoactive intestinal peptide (VIP) and parathyroid hormone (PTH) using preparations of cerebral microvessels and arteries. Cerebral microvessels obtained from rats, guinea‐pigs, cattle, and pigs all responded potently to bovine (b) PTH‐(1‐34), whereas considerable between‐species variability was observed in the responsiveness to VIP. The homologous peptide to VIP, PHI (porcine heptacosapeptide), stimulated adenylate cyclase in both rat microvessels and a broken‐cell preparation of bovine arteries. The ED50 values for activation of bovine arterial adenylate cyclase by VIP, PHI, and bPTH‐(1‐34) were 6.9 nM, 10 nM, and 100 nM, respectively, with the following order of efficacy: VIP = PHI > bPTH‐(1‐34). The other related peptides, hpGRF (human pancreatic growth hormone releasing factor), secretin, and glucagon, and the fragment VIP‐(10‐28) were inactive. The PTH antagonist, [Nle8, Nle18, Tyr34]bPTH‐(3‐34) amide, inhibited bPTH‐(1‐34) activation of vascular adenylate cyclase but did not affect activation by VIP using either microvessels or arteries. VIP or PHI demonstrated an additive effect with bPTH‐(1‐34) on vascular adenylate cyclase activity. However, the effects of VIP and PHI were nonadditive with each other. These data suggest that VIP and bPTH‐(1‐34) activate cerebral vascular adenylate cyclase by interacting with pharmacologically distinct receptors, whereas PHI and VIP likely interact with a common receptor.
Concanavalin A Binding Glycoproteins in Subcellular Fractions from the Developing Rat Cerebral Cortex
Authors: Rudge John S. , Murphy Sean
Published: December 2006

Abstract: Synaptic plasma membrane (SPM) and mitochondrial fractions were prepared from 3‐50‐day rat cerebral cortex and their purity assessed. The fractions were subjected to electrophoresis on slab gels, stained for protein, and overlaid with 125I‐concanavalin A (ConA). ConA binding glycoproteins (CABGs) were revealed by autoradiography. In the SPM fraction CABGs of MW 25,000, 63,000, 80,000, 115,000, 174,000, and 239,000 increased while those of MW 47,000, 75,000, and 190,000 decreased developmentally. In the mitochondrial fraction, CABGs of MW 25,000, 44,000, 115,000 and 174,000 increased while those of 34,000, 43,000, 47,000, 51,000, 80,000, 107,000, and 195,000 decreased develop‐mentally. CABGs of MW 32,000, 63,000, 88,000, 153,000, 190,000, and 239,000 appear to be unique to the SPM fraction and those of MW 34,000, 107,000, and 195,000 are unique to the mitochondrial fraction.
Cross‐Reaction of Anti‐Rat B‐50: Characterization and Isolation of a “B‐50 Phosphoprotein” from Bovine Brain
Authors: Oestreicher Anna Beate , van Duin Marcel , Zwiers Henk , Gispen Willem Hendrik
Published: December 2006

Abstract: Antibodies to the phosphoprotein B‐50 of rat brain were used to trace cross‐reacting brain proteins of vertebrates. With the SDS‐gel‐immunoperoxidase method, a cross‐reacting protein (CP) of apparent Mr 53,000 was demonstrated in the homogenate and the synaptic plasma membrane fraction of bovine brain. Sequence 1–24 of adrenocorticotropin (ACTH1‐24) (10−5M and 10−4M) inhibited endogenous phosphorylation of CP in synaptic plasma membranes. The protein was partially characterized and purified to homogeneity from bovine brain by procedures previously described for rat B‐50. CP was enriched in ammonium sulfate precipitated protein (ASP) fractions and phosphorylated by an endogenous protein kinase. Two‐dimensional gel analysis of bovine and rat ASP showed that the cross‐reacting protein had an isoelectric point less acidic than B‐50. Limited proteolysis by Staphylococcus aureus protease yielded a “peptide map” analogous to B‐50. Two major fragments of Mr 30,000 and 17,000 were produced. In addition, CP exhibited other similarities to rat B‐50: phosphorylation by rat brain protein kinase C, microheterogeneity observed after isoelectric focusing, and possibly degradation by endogenous proteolysis. Cross‐reaction of proteins in brain homogenates of other mammalian species and of chicken was demonstrated: the Mr of the proteins ranged from 47,000 to 53,000. We conclude that (1) the cross‐reacting bovine protein is a “B‐50 protein,” and (2) the Mr of the “B‐50 protein” varies from species to species.