bluuebunny/crossref_metadata_2025 huggingface dataset:
Cholinergic‐Adrenergic Receptor Interactions in Cerebral Microvessels
Authors: Grammas P. , Diglio C. A. , Giacomelli F. , Wiener J.
Published: October 2006

Enriched capillary preparations isolated from rat cerebral cortex were used to evaluate cholinergic‐adrenergic receptor interactions in cerebral endothelium. Possible receptor interactions were determined by measuring an intracellular mediator, cyclic AMP and alterations in GTP‐sensitive agonist binding. Unstimulated microvessel homogenates generate 66 ± 16 pmol/mg/10 min of cyclic AMP. Adrenergic agonists norepinephrine and isoproterenol increase cyclic AMP to 147 ± 31 and 149 ± 23 pmol/mg/10 min, respectively. Addition of the muscarinic agonist carbachol has no effect on basal cyclic AMP but it completely blocks the stimulation elicited by adrenergic agonists. The displacement of quinuclidinyl benzilate (QNB) by carbachol yields an IC50 of 1.5 ± 0.45 x 10−4M and a Hill coefficient of 0.54 ± 0.07, indicating a heterogeneous population of binding sites. Guanine nucleotides shift the displacement curve to the right (IC50, 4.7 ± 0.16 x 10−4M) and convert the binding site population to greater homogeneity (0.76 ± 0.18). Isoproterenol prevents both the affinity shift and binding site conversion evoked by guanine nucleotides. These data suggest that cholinergic‐adrenergic interactions occur at both the level of receptor binding and the generation of an intracellular messenger. Since cyclic AMP has been purported to play a role in regulation of blood–brain barrier permeability, the existence of adrenergic‐cholinergic, i.e., excitatory‐inhibitory modulators of adenylate cyclase in cerebral endothelium, suggests that these receptors may mediate physiological and/or pathological alterations of cerebrovascular permeability.
Distribution of Guanine Deaminase in Mouse Brain
Authors: Berger Sosamma J. , Carter Joyce G. , Lowry Oliver H.
Published: October 2006

Guanine deaminase was measured in nearly 100 different areas of mouse brain. The levels are relatively high in all parts of the telencephalon, both gray and white. It is especially active in parts of the olfactory tubercle and amygdala. Levels in the diencephalon range from low to as high as in the telencephalon. Brain areas caudal to the diencephalon, including all parts of the cerebellum, are almost uniformly below the level of detection. The enzyme is also virtually absent from the retina. The extreme range of concentration suggests that guanine deaminase might play a role in the metabolism of a neuroeffector.
Studies on a Cerebellar 50,000‐Dalton Protein Associated with Cerebellar Hypoplasia in Jaundiced Gunn Rats: Its Identity with Glial Fibrillary Acidic Protein as Evidenced by the Improved Immunoblotting Method
Authors: Aono Sachiko , Sato Hiroshi , Semba Reiji , Kashiwamata Shigeo , Eng Lawrence F.
Published: October 2006

On the basis of our previous findings that a 50,000‐dalton protein (GR‐50) shows a marked increase in the hypoplastic cerebellum of jaundiced homozygous Gunn rats and its electrophoretic behavior is similar to that of glial fibrillary acidic protein (GFAP), we tried to identify GR‐50 as GFAP by two‐dimensional electropho‐resis of rat cerebellar membrane proteins using an improved immunoblotting method. In this method a blot immunostained for a specific antigen was also visualized for other proteins, thereby enabling us to determine the location of the antigen on the blot more precisely. With the methodology it was found that GFAP antigen occupied exactly the same position as GR‐50 on the blot, suggesting strongly the identity of both proteins. Immunohistochemical studies revealed that in the cerebellum of homozygotes compared with that of heterozygotes GFAP antigen was greatly increased and that it was especially rich in the homozygous rat cerebellar lobules with a high degree of hypoplasia. Further, it was shown that not only the fibers of the Bergmann glial cells but also their somata were intensely immunostained in the affected lobules. A 47,000‐dalton protein (SG‐47), which has been reported to be increased in staggerer mutant mice with cerebellar hypoplasia, also immunoreacted with the antiserum to GFAP.
Complete Conversion of Brain D2 Dopamine Receptors from the High‐ to the Low‐Affinity State for Dopamine Agonists, Using Sodium Ions and Guanine Nucleotide
Authors: Grigoriadis Dimitri , Seeman Philip
Published: October 2006

Since previous work had shown that brain D2 3,4‐dihydroxyphenylethylamine (dopamine) receptors were only partly converted from their high‐affinity state to their low‐affinity state, we here tested whether it was possible to obtain a complete 100% conversion of these receptors into their low‐affinity state. It was first essential to resolve the components of [3H]spiperone binding to dopaminergic sites and nondopaminergic sites in rat striatal homogenates. In the presence of 50 μMS‐sulpiride (to occlude the dopaminergic sites), therefore, we first determined that the residual binding of [3H]spiperone (approximately 20%) was inhibited by serotonergic agonists much more effectively than dopamine or noradrenaline, thus identifying the serotonergic component of [3H]spiperone binding. Thus, dopamine (or ADTN) inhibited the binding of [3H]spiperone at a high‐affinity site (with dissociation constant of 10 nM dopamine), at a low‐affinity site (with dissociation constant of 2,000 nM dopamine), and at the serotonergic site (with dissociation constant of 50,000 nM dopamine). In the absence of sodium ions, the high‐affinity site was about 50% occupied by [3H]spiperone, and guanine nucleotide had no effect on this proportion. In the presence of 120 mM NaCl, however, the high‐affinity site was reduced to 15% and guanine nucleotide completely eliminated this high‐affinity site, 100% of the sites having been completely converted to their low‐affinity state. Using [3H]N‐propyl‐norapomorphine to label the high‐affinity state of the dopamine receptor, 50% conversion into the low‐affinity state occurred at 45 mM NaCl, 69 mM NaCl, and 202 mM KCl. We conclude that it is possible to convert brain D2 dopamine receptors completely into their low‐affinity state, in the presence of NaCl and a guanine nucleotide, providing that appropriate allowance is made for the serotonergic component of [3H]spiperone binding.
Possible Allosteric Interaction of 4‐Aminopyridine with Rat Brain Muscarinic Acetylcholine Receptors
Authors: Lai Wi S. , Ramkumar Vickram , El‐Fakahany Esam E.
Published: October 2006

The interaction of the potassium channel blocker 4‐aminopyridine (4‐AP) and its analogs with muscarinic acetylcholine receptors was studied in rat brain homogenate. 4‐AP displaced specific [3H]quinuclidinyl benzilate ([3H]QNB) binding in a concentration‐dependent fashion. Hill coefficient values decreased with increasing the concentration of [3H]QNB and different analogs of 4‐AP demonstrated varying potencies. Scatchard analysis of saturation isotherms of specific [3H]QNB binding showed that low concentrations of 4‐AP slightly reduced maximum binding without affecting the equilibrium dissociation constant, whereas higher concentrations reduced maximum binding further and significantly increased the equilibrium dissociation constant. Schild plots of these data resulted in curvilinear functions. The results are discussed in terms of possible allosteric interactions between potassium channels and muscarinic receptor binding sites.
Somatostatin Precursor in the Rat Striatum: Changes After Local Injection of Kainic Acid
Authors: Sperk Günther , Widmann Rudolf
Published: October 2006

Abstract: The molecular forms of somatostatin contained in the rat striatum were separated by size‐exclusion HPLC. Three major peaks of somatostatin‐like immunoreactivity (SLI) were resolved. Two peaks cochromatographed with synthetic somatostatin‐14 (SS‐14) and somatostatin‐28 (SS‐28), respectively. One peak exhibited a higher molecular weight (about 10,000) and may contain a proform of somatostatin. Local injection of the neurotoxin kainic acid (1 μg) into the left striatum resulted in a persistent decrease (65–85%) of all three forms of somatostatin. In the contralateral—not injected—striatum a decrease of SLI was also observed which was maximal (45%) after 2 days and was largely abolished after 7 days. This decrease of SLI in the contralateral striatum, however, was due mainly to a decrease of SS‐14 and SS‐28 but not of the putative proform. Our data suggest that kainic acid causes a destruction of somatostatin‐containing perikarya in the injected striatum, whereas in the contralateral striatum increased release with subsequent inactivation of SS‐14 and SS‐28 takes place. The putative somatostatin proform may serve as neurochemical marker for somatostatin‐containing perikarya in the striatum.
Solubilization of Membrane‐Bound Acetyicholinesterase by a Phosphatidylinositol‐Specific Phospholipase C
Authors: Futerman Anthony H. , Low Martin G. , Michaelson Daniel M. , Silman Israel
Published: October 2006

Abstract: Phosphatidylinositol‐specific phospholipase C (PIPLC) quantitatively solubilizes acetylcholinesterase (AChE) from purified synaptic plasma membranes and intact synaptosomes of Torpedo ocellata electric organ. The solubilized AChE migrates as a single peak of sedimentation coefficient 7.OS upon sucrose gradient centrifugation, corresponding to a subunit dimer. The catalytic subunit polypeptide of AChE is the only polypeptide detectably of soubilized by PIPLC. This selective removal of AChE does not affect the amount of acetylcholine released from intact synaptosomes upon K+ depolarization. PIPLC also quantitatively solubilizes AChE from the surface of intact bovine and rat erythrocytes, but only partially solubilizes AChE from human and mouse erythrocytes. The AChE released from rat and human erythrocytes by PIPLC migrates as a ∼ 7S species on sucrose gradients, corresponding to a catalytic subunit dimer. PIPLC does not solubilize particulate AChE from any of the brain regions examined of four mammalian species. Several other phospholipases tested, including a nonspecific phospholipase C from Clostridium welchii, fail to solubilize AChE from Torpedo synaptic plasma membranes, rat erythrocytes, or rat striatum.
Molecular Properties of a Cholinergic Differentiation Factor from Muscle‐Conditioned Medium
Authors: Weber Michel J. , Raynaud Brigitte , Delteil Christine
Published: October 2006

Abstract: Conditioned medium by a variety of rat nonneuronal cells contains a protein involved in the differentiation of cholinergic neurons in cultures prepared from newborn rat superior cervical ganglion, from nodose ganglion, and from embryonic spinal cord. We have determined some hydrodynamic properties of this factor using as a bioassay the increase in choline acetyltransferase activity in sympathetic neurons grown for 12–15 days in the presence of the factor. The Stokes' radius, measured by molecular sieving chromatography on an Ultrogel AcA 44 column, was similar to that of ovalbumin (27.6 Å). By analysis on 5–20% linear sucrose gradients made in H2O and D2O, we determined the partial specific volume (0.68 ml × g−1) and the sedimentation coefficient (2.1S). These data allowed the calculation of the molecular weight (21,000) and the frictional ratio f/f0 (1.56). The elution pattern of the factor from a SynChropak CM 300 HPLC cation exchange column suggested that it was a basic protein. The activity of this factor was unaffected by heat treatment at 100°C for 10 min.
Changes in In Vitro Brain and Spinal Cord Protein Phosphorylation After a Single Oral Administration of Tri‐o‐Cresyl Phosphate to Hens
Authors: Patton Suzanne E. , Lapadula Daniel M. , O'Callaghan James P. , Miller Diane B. , Abou‐Donia Mohamed B.
Published: October 2006

Abstract: The effect of a single oral 750 mg/kg dose of tri‐o‐cresyl phosphate (TOCP) on the endogenous phosphorylation of brain and spinal cord proteins was assessed in hens during the development of and recovery from delayed neurotoxicity. Crude membrane and cytosolic fractions were prepared from the brains and spinal cords of control and TOCP‐treated hens at 1, 7, 14, 21, 35, and 55 days after treatment. Brain and spinal cord protein phosphorylation with [γ‐32P]ATP was analyzed by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE), autoradiography, and microdensitometry. TOCP administration conferred calcium and calmodulin dependence on the phosphorylation of a few brain cytosolic proteins and caused an increase in the phosphorylation of a number of other cytosolic and membrane proteins. This effect of TOCP was large in magnitude, and its time course reflected the onset of and recovery from the signs of ataxia and paralysis associated with delayed neurotoxicity in the hen. The molecular weights (Mr) and maximal phosphorylation (percent of control) for the most prominently affected bands were as follows: brain cytosol—50K (183%), 55K (575%), 60K (529%), 65K (273%), and 70K (548%); brain membranes—50K (622%) and 60K (697%); and spinal cord cytosol—20K (182%). The role of endogenous phosphorylation reactions in and their potential usefulness as biochemical indicators of delayed neurotoxicity are being explored further.
Structural and Functional Characteristics of Insulin Receptors in Rat Neuroblastoma Cells
Authors: Heidenreich Kim A. , Gilmore Patricia R.
Published: October 2006

Abstract: Insulin receptors were detected in a variety of rat neuroblastoma and glioma cell lines. The binding of 125I‐insulin to B103 neuroblastoma cells had characteristics typical of insulin receptors in other tissues, including high affinity for insulin, low affinity for insulin‐like growth factor I (IGF‐I), and curvilinear Scatchard plots. Using photoaffinity labeling procedures and sodium dodecyl sulfate (SDS) gel electrophoresis to analyze the subunit structure of insulin receptors in B103 cells, the predominantly labeled protein had an apparent molecular weight of 125K and the mobility of this protein was shifted after removal of sialic acid residues. On the basis of size and susceptibility to neuraminidase, the insulin binding subunit in neuroblastoma cells was identical to the α‐subunit of insulin receptors in adipocytes and different from the 115K subunit found in brain. The presence of an “adipocyte′’ form of the insulin receptor in clonal cells derived from brain is probably a consequence of transformation and results from more extensive oligosaccharide processing of the 115K receptor expressed in normal brain cells. The fully glycosylated receptors in neuroblastoma cells were capable of exerting functions typical of insulin receptors in adipocytes such as internalization of insulin and stimulation of glucose transport.
Ethanol's Effects on Cortical Adenylate Cyclase Activity
Authors: Saito Toshikazu , Lee John M. , Tabakoff Boris
Published: October 2006

Abstract: The effects of ethanol on β‐adrenergic receptor‐coupled adenylate cyclase (AC) of mouse cerebral cortex were examined. The addition of ethanol (20–500 mM) to incubation mixtures containing cortical membranes demonstrated that ethanol could increase AC activity and potentiate the stimulatory effects of guanylylimidodiphosphate [Gpp(NH)p] on AC activity. Ethanol increased the rate of activation of AC by guanine nucleotides and concomitantly decreased the EC50 for magnesium required to achieve maximal stimulation of cortical AC. The EC50 values for Gpp(NH)p and isoproterenol stimulation of AC activity were also altered by ethanol. Ethanol was capable of stimulating AC extracted by use of digitonin. The AC activity in the digitonin extract was no longer sensitive to the addition of Gpp(NH)p or NaF, but was still stimulated by ethanol. We propose multiple sites of action for ethanol in stimulating cortical AC activity. These sites include actions at the β‐adrenergic receptor, at the G/F coupling proteins, and at the catalytic unit of cortical AC. Comparison of ethanol's actions on cortical β receptor coupled AC activity with prior reported actions of ethanol on striatal dopamine (DA)‐sensitive AC indicated differential sensitivities of these two AC systems to ethanol. These differences may be determined by specific coupling characteristics of the striatal and cortical AC systems or by differences in the plasma membranes in which striatal and cortical AC systems are located.
Rapid Sequestration and Degradation of Somatostatin Analogues by Isolated Brain Microvessels
Authors: Pardridge William M. , Eisenberg Jody , Yamada Tadataka
Published: October 2006

Abstract: Somatostatin (SRIF) is a putative peptide neurotransmitter that may interact with brain capillaries following neurosecretion of the peptide. The present studies investigate the binding and metabolism of SRIF analogues in isolated bovine brain microvessels. 125I [Tyr1]SRIF was rapidly degraded by capillary aminopeptidase with a half‐time of approximately 3 min at 23°C. The microvessel aminopeptidase had a low affinity and high capacity for the peptide, Km= 76 μM and Vmax= 74 nmol min−1. 125I‐[Tyr11]SRIF was converted to free iodotyrosine at a much slower rate, presumably by a lower‐activity endopeptidase. 125I‐[Tyr 11]SRIF was rapidly bound by microvessels, whereas another basic peptide, [Tyr8]bradykinin, or an acidic peptide, CCK8, or a neutral peptide, leucine enkephalin, were bound to a considerably less extent. The binding of 125I‐[Tyr11]SRIF to the capillaries was nonsaturable up to a concentration of 1 μg/ml of unlabeled peptide, and the binding reaction was extremely rapid, reaching equilibrium within 5 s at either 0°C or 37°C. Approximately 20% of the SRIF bound by the microvessels was resistant to acid wash and presumably represented internalized peptide. In addition, the 125I‐[Tyr11]SRIF bound rapidly to the endothelial cytoskeleton remaining after a 1% Triton X‐100 extraction of the microvessels. The peptide‐cytoskeletal binding reaction was nonsaturable up to 1 μg/ml of unlabeled [Tyr11]SRIF, but it was inhibited by 0.5% polylysine or 0.8 M KC1 and was stimulated by 1 mM dithiothreiotol. These studies suggest that brain microvessels rapidly sequester and degrade SRIF analogues and that this may represent one mechanism for rapid inactivation of the neuropeptides subsequent to neurosecretion.
Ganglioside GM1 Does Not Initiate, but Enhances Neurite Regeneration of Nerve Growth Factor‐Dependent Sensory Neurones
Authors: Doherty Patrick , Dickson John G. , Flanigan Thomas P. , Walsh Frank S.
Published: October 2006

Abstract: An enzyme‐linked immunoadsorbent assay (ELISA) for neurofilament protein was utilised to quantify the effect of exogenous ganglioside on neurite regeneration in cultures of dorsal root ganglion neurones. In contrast to nerve growth factor (NGF), ganglioside GM1 (100 μg/ml) failed to support neuronal survival and neurite regeneration as quantified by the ELISA assay and confirmed by morphological criteria. However, the simultaneous presence of GM1 (100 μg/ml) and NGF (0.5–5 ng/ml) throughout a 5‐day period of culture resulted in an enhancement of previously reported NGF‐induced increases in the expression of neurofilament protein. Further, the addition of GM1 (0–200 μg/ml) at 48 h in vitro to cultures initially established in the presence of 5 ng/ml NGF substantially increased the subsequent expression of neurofilament protein, this response being both independent of and not potentiated by NGF. The results in the present system suggest that GM1 cannot initiate a programme of neurite regeneration; however, GM1 can enhance this process with the response being secondary to the effect of NGF.
Effects of Rat Ovariectomy on CSF Monoamine Metabolite Levels and Elimination
Authors: Duval Christine , Mignot Emmanuel
Published: October 2006

Abstract: Cerebrospinal fluid (CSF) was removed from the third ventricle of anesthetized male, female, and ovariectomized rats. CSF 3,4‐dihydroxyphenylethylamine and serotonin metabolite levels [dihydroxyphenylacetic acid, homovanillic acid (HVA), and 5‐hydroxyindoleacetic acid (5‐HIAA)] were determined on 15‐min samples by liquid chromatography coupled with electrochemical detection. Monoamine oxidase inhibition was used for studying metabolite turnover in the CSF. No difference was observed between male, ovariectomized, and sham‐operated female rats. However, ventricular CSF HVA and 5‐HIAA levels were significantly higher in the ovariectomized than in the sham‐operated rats. These differences do not reflect effects of ovariectomy on brain metabolite production but indicate slower metabolite elimination from the CSF.
Cyclic AMP Concentrations in Rat Neocortex and Hippocampus During and Following Incomplete Ischemia: Effects of Central Noradrenergic Neurons, Prostaglandins, and Adenosine
Authors: Blomqvist Photjanee , Lindvall ROlle , Stenevi Rulf , Wieloch Tadeusz
Published: October 2006

Abstract: The concentrations of cyclic AMP, noradrenaline, glycogen, glucose, lactate, pyruvate, labile phosphate compounds, and free fatty acids were investigated in the rat neocortex and hippocampus during and following cerebral ischemia. An incomplete ischemia of 5 and 15 min duration was induced by bilateral carotid clamping combined with hypotension. The postischemic events were studied after 5, 15, and 60 min of recirculation. Five minutes of ischemia did not significantly alter the neocortical or hippocampal concentrations of cyclic AMP. After 15 min of ischemia the neocortical levels decreased significantly below control values. In the recirculation period following ischemia a significant elevation of the cyclic AMP concentrations was observed. Following 5 min of recirculation after 5 min of ischemia the levels increased from 2.53 ± 0.21 nmol ± g−1 to 5.18 ± 0.09 nmol ± g−1 in the neocortex and from 2.14 ± 0.16 nmol ± g−1 to 3.52 ± 0.35 nmol ± g−1 in the hippocampus. Five minutes of recirculation following 15 min of ischemia led to a significant increase in the levels of cyclic AMP, to 12.86 ± 1.43 nmol ± g−1 in the neocortex to 5.58 ± 0.57 nmol ± g−1 in the hippocampus. With longer recirculation periods the cyclic AMP levels progressively decreased and were similar to control values after 60 min. Depletion of cortical noradrenaline by at least 95% was performed by injections of 6‐hydroxydopamine into the ascending axon bundles from the locus ceruleus. The lesion did not significantly change the ischemic or postischemic neocortical and hippocampal levels of cyclic AMP, glycogen, or free fatty acids including arachidonic acid. Treatment of the animals with theophyllamine (23, 46, and 92 mg ± kg−1) or indomethacin (10 mg ± kg−1) did not affect the postischemic levels of cyclic AMP. It is concluded that central noradrenergic neurons, prostaglandins, and adenosine are not of major importance for the observed postischemic elevations of cyclic AMP and that the changes in the concentrations of free fatty acids measured during and following ischemia are not mediated by noradrenergic neurons.
Purification and Characterization of Four NADPH‐Dependent Aldehyde Reductases from Pig Brain
Authors: Cromlish James A. , Yoshimoto Carol K. , Flynn T. Geoffrey
Published: October 2006

Abstract: By a procedure involving ammonium sulfate precipitation, gel filtration, and affinity chromatography, four aldehyde reductases (ALRs) were purified to enzymatic homogeneity from pig brain. These enzymes, designated ALR1, ALR2, ALR3, and succinic semialdehyde reductase were chemically and physically identical with, respectively, the high‐Km aldehyde reductase, the low‐Km aldehyde reductase, carbonyl reductase, and succinic semialdehyde reductase of other tissues and species. The purification procedure allows the purification of these enzymes from the same tissue homogenate in amounts sufficient for characterization and other enzymatic studies. This methodology should be applicable to the simultaneous and rapid purification of aldehyde reductases from other tissues.
Catabolism of Exogenous and Endogenous Sphingomyelin and Phosphatidylcholine by Homogenates and Subcellular Fractions of Cultured Neuroblastoma Cells. Effects of Anesthetics
Authors: Mooibroek Marilyn J. , Cook Harold W. , Clarke Joe T. R. , Spence Matthew W.
Published: October 2006

Abstract: Cultured murine neuroblastoma cells contain a neutral, Mg2+‐stimulated sphingomyelinase and an alkaline phosphatidylcholine‐hydrolyzing activity that are enriched in the plasma membrane fraction. The reaction products of sphingomyelin catabolism are phosphocholine and ceramide and those of phosphatidylcholine, glycerophosphocholine and fatty acid. These reactions were studied with endogenous as well as exogenous liposomal substrates. With both exogenous and endogenous substrates, the sphingomyelinase activity was stimulated two‐to threefold by Mg2+ and a further three‐to fourfold by volatile anesthetic agents. Stimulation was concentration‐dependent and corresponded to anesthetic potency: methoxyflurane > halothane > enflurane. Greater than 80% of the plasma membrane sphingomyelin was hydrolyzed within 2 h in the presence of Mg2+ and anesthetic. In contrast, the activity with exogenous and endogenous phosphatidylcholine was unaffected by Mg2+ or Ca2+ and was markedly inhibited (50–80%) by anesthetic agents. The degree of inhibition was concentration‐dependent and corresponded to anesthetic potency. The quantitative importance of choline‐containing lipids in cell membranes, the relatively exclusive localization of the neutral Mg2+ ‐stimulated sphingomyelinase in cells of neural origin, the totally different type of hydrolytic attack on phosphatidylcholine, and the reciprocal effects of anesthetics on the hydrolysis of these two lipids strongly suggest important roles for these activities in cell membranes in general and in the neuron in particular.
Effects of Undernutrition and Thyroid State on the Ontogenetic Changes of D1, D2, and D3 Brain‐Specific Proteins in Rat Cerebellum
Authors: Patel Ambrish J. , Hunt Anthony , Meier Eddi
Published: October 2006

Abstract: Disturbances in metabolic balance brought about by alterations in thyroid state and undernutrition during early life had a marked effect on the concentrations of the brain‐specific proteins, D1, D2, and D3 in the developing rat cerebellum. In normal rats, the concentrations of D1 and D3 increased and that of D2 decreased during the first 3 weeks after birth. In the hyperthyroid state a small but consistent advancement was observed in the developmental curves of these proteins. The hypothyroid state caused a marked retardation in the maturational pattern of D1 and D2 but not of D3. In undernutrition, at 6 days the concentrations of D1 and D3 proteins were higher than in controls, but thereafter the developmental increase was markedly delayed for D1 only. The concentration of D2 was normal at 6 days, but after the first week a marked retardation was observed in the maturational pattern of this protein in undernourished rats. In addition, the “anodic‐immature”form of D2 predominated in 6‐day‐old controls, but this was gradually replaced by a “cathodic‐mature”form which progressively became the dominant form of D2 in 35‐day‐old rat cerebellum. The developmental switch in terms of the two forms was also advanced in hyperthyroidism and retarded in thyroid deficiency and undernutrition. Furthermore, daily treatment of hypothyroid rats with physiological doses of thyroxine from birth restored the concentrations of D1 and D2 to normal, but that of D3 was increased above control levels, indicating differences between the proteins in their sensitivity to mechanisms of control by thyroid hormone. Also, the overall effects of undernutrition were markedly different from those of hypothyroidism.
Neurochemical Profile of Lu 19‐005, a Potent Inhibitor of Uptake of Dopamine, Noradrenaline, and Serotonin
Authors: Hyttel John , Larsen Jens‐Jørgen
Published: October 2006

Abstract: The neurochemical profile of a new compound, Lu 19–005 [(±)trans‐3‐(3,4‐dichlorophenyl)‐N‐methyl‐1‐indanamine hydrochloride], has been investigated. Lu 19–005 is a potent inhibitor of the synaptosomal uptake of 3,4‐dihydroxyphenylethylamine (dopamine, DA), noradrenaline (NA), and 5‐hydroxytryptamine (5‐HT, serotonin). In this respect it resembles diclofensine, whereas compounds such as GBR 13.069 and bupropion are more selective DA‐uptake inhibitors. Although Lu 19–005 releases DA in in higher concentrations it must be considered as an uptake inhibitor, as the accumulation of DA is inhibited in much lower concentrations. Lu 19–005 attenuates the DA and NA depletion caused by 6‐hydroxydopamine in mouse brain. These properties confirm the DA‐ and NA‐uptake‐inhibitory properties of the compound. In receptor‐binding models and functional in vitro tests Lu 19–005 is devoid of dopaminergic‐, serotonergic‐, noradrenergic‐, histaminergic‐, and cholinergic‐inhibiting properties. Since DA, NA, and 5‐HT seem to be involved in depression, the profile of Lu 19‐005–with equally potent activity on the three neuronal systems–makes it an interesting experimental tool and a potential new antidepressant agent.
Leptinotoxin‐h Action in Synaptosomes and Neurosecretory Cells: Stimulation of Neurotransmitter Release
Authors: Madeddu Luisa , Saito Isamu , Hsiao Ting H. , Meldolesi Jacopo
Published: October 2006

Abstract: Guinea pig brain cortex synaptosomes and neurosecretory PC12 cells were loaded with [3H]3,4‐di‐hydroxyphenylethylamine ([3H]DA, [3H]dopamine) and then exposed to leptinotoxin‐h (LPTx) (purified and partially purified preparations, obtained from the hemo‐lymph of Leptinotarsa haldemani). In a Ca2+‐containing Ringer medium the toxin induced prompt and massive release of the neurotransmitter. Half‐maximal effects were obtained at concentrations estimated of approximately 3 × 10−11M for synaptosomes, and 1.5 × 10−10M for PC12 cells. Release responses in the two experimental systems investigated were dependent to different extents on the Ca2+ concentration in the medium. In synaptosomes clear, although slow, release of [3H]DA was elicited by the toxin even in Ca2+‐free, EGTA‐containing medium, provided that high (in the 10−10M range) concentrations were used; near‐maximal responses were observed at 10−5M Ca2+. In contrast, the toxin‐induced release from PC12 cells was appreciable only at 3 × 10−5M Ca2+, and was maximal at 2 × 10−4M and above. In both synaptosomes and PC12 cells Sr2+ and Ba2+ could substitute for Ca2+; Co2+ was inhibitory, whereas Mn2+ failed to modify the release induced by the toxin in Ca2+‐containing medium. Organic blockers of the voltage‐dependent Ca2+ channel (verapamil and nitrendipine) and calmodulin blocking drugs (trifluoperazine and calmidazolium) failed to inhibit the toxin‐induced release of [3H]DA. LPTx induced profound morphological effects. Synaptosomes treated in the Ca2+‐containing medium exhibited fusion of synaptic vesicles, formation of numerous infoldings and large cisternae, and alterations of mitochondria. In the Ca2+‐free medium the effects were similar, except that their appearance was delayed, and mitochondria were well preserved. Swelling was observed in PC12 cells, accompanied by enlargement of the Golgi area, accumulation of multivesicular bodies, mitochondrial alterations, and decreased number of secretion granules (Ca2+‐containing medium). Morphometric analyses revealed a good correlation between the decrease of both synaptic vesicles (synaptosomes) and neurosecretory granules (PC12 cells), and the release of [3H]DA measured biochemically. This is a good indication that the release effect of the toxin is due to stimulation of exocytosis. Taken as a whole, these results confirm the similarity of the effects of LPTx with α‐latrotoxin of the black widow spider venom, mentioned in the companion article. However, differences in effect and target specificity suggest that the two toxins are specific to separate binding sites. Because of its profound and exclusively presynaptic effects LPTx promises to be an interesting tool for the study of the regulation of exocytotic neuro‐transmitter release.
Retrograde Axonal Transport of Phospholipid in Rat Sciatic Nerve
Authors: Bisby Mark A.
Published: October 2006

Abstract: Retrograde axonal transport of phospholipid was studied in rat sciatic motoneuron axons by placing collection crushes on the nerve at intervals after injection of [methyl‐3H]choline into the lumbosacral spinal cord, and allowing labelled material undergoing anterograde or retrograde movement to accumulate adjacent to the collection crushes. Control experiments showed that the accumulations of label were not a result of local uptake of circulating precursor. The majority of the 3H label was associated with phosphatidylcholine. Accumulation of label at the distal collection crush, representing retrograde transport, was observed subsequent to the anterograde transport of phospholipid. In comparison with previous study on retrograde transport of protein, the following points were noted: (1) onset of retrograde transport occurred at approximately the same time after precursor injection (10–20 h) for both protein and phospholipid; (2) retrograde transport of lipids was more prolonged: maximum retrograde transport occurred later for phospholipid (30 h) than for protein (15–20 h), and declined to half‐maximum between 49 and 99 h, compared to a corresponding value of 24–28 h for protein; (3) the proportion of total anterograde‐transported activity subsequently undergoing retrograde transport was less in the case of phospholipid, at least over the time interval studied (up to 99 h after precursor injection). The similar times of onset of retrograde transport of phospholipid and protein support the concept of retrograde transport as a recycling mechanism returning to the cell body membrane fragments that were earlier transported into the axon. Coordinated retrograde transport of labelled protein and phospholipid components of the recycled membranes would be predicted. Differences between protein and phospholipid in the subsequent time course and amount of retrograde transport may reflect differences in axonal handling of protein and lipid. Both the more prolonged outflow of labelled lipids from cell body into axon and exchange with a distal pool of unlabelled phospholipid may account for the prolonged time course of retrograde transport of labelled lipid.
Phosphatidylinositol:myo‐Inositol Exchange Activity in Intact Nerve Endings: Substrate and Cofactor Dependence, Nucleotide Specificity, and Effect on Synaptosomal Handling of myo‐Inositol
Authors: Berry Gerard , Yandrasitz John R. , Cipriano Victoria M. , Hwang Shing Mei , Segal Stanton
Published: October 2006

Abstract: Micromolar concentrations of CMP produced a large increase in Mn2+ ‐dependent phosphatidylinositol:myo‐inositol exchange activity in isolated nerve endings or synaptosomes. The apparent Km for CMP was 2 μM, and that for myo‐inositol was 38 μM. Only cytidine nucleotides were capable of enhancing activity, and this effect is probably specific for CMP, because the synaptosomal preparation rapidly converted CTP or CDP to CMP. Manganese did not affect the uptake of myo‐inositol into the synaptosomal cytosolic fraction or myo‐inositol levels. Determinations of myo‐inositol specific activity showed that the Mn2+‐enhanced labeling of phosphatidylinositol was not accompanied by a decrease of label content in free myo‐inositol. This lack of an effect on intrasynaptosomal myo‐inositol and the dependence of exchange on cytidine nucleotides whereas cytidine itself was previously found to be without effect show that for the bulk of Mn2+‐dependent exchange activity, it is the myo‐inositol in the incubation medium that is being directly incorporated into membrane‐bound phosphatidylinositol. Because CMP dependence is the hallmark of exchange catalyzed by CDP‐diacylglycerol:inositol phosphatidyl transferase, this enzyme is likely to be responsible for most of the exchange activity in synaptosomes. The strong affinity of this exchange system for CMP suggests that endogenous levels of this nucleotide might support Mn2+‐dependent exchange in the absence of added nucleotide.
Effect of Various Centrally Acting Drugs on the Efflux of Dopamine Metabolites from the Rat Brain
Authors: Westerink B. H. C. , Kikkert R. J.
Published: October 2006

Abstract: The influence of chlorpromazine, haloperidol, morphine, chloral hydrate, γ‐butyrolactone, probenecid, kainic acid, oxotremorine, pargyline, yohimbine, (+)‐amphetamine, and cocaine on the efflux rate of 3,4‐dihydroxyphenylacetic acid (DOPAC) from four brain areas was studied. All drugs studied except pargyline and morphine had an effect on the transport of DOPAC and homovanillic acid (HVA) from the brain. Nine drugs inhibited the efflux of DOPAC and HVA, whereas (+)‐amphetamine stimulated this transport. These data suggest that most centrally acting drugs can interfere with the elimination of 3,4‐dihydroxyphenylethylamine (DA or dopamine) metabolites from the brain. These effects are heterogeneously distributed throughout the brain and are probably related to indirect nonspecific drug effects. This implies that drug‐induced changes in DA metabolite concentrations, especially when these changes are slight to moderate, cannot directly be translated to changes in the production rate of these metabolites. By studying five control groups, we concluded that formation and transport of DOPAC are not synchronized in the various brain areas.
Vesicular Storage of 3,4‐Dihydroxyphenylethylamine and Noradrenaline in Terminal Sympathetic Nerves of Dog Spleen and Kidney
Authors: Muller B. D. , Bell C.
Published: October 2006

Abstract The subcellular distribution of 3,4‐dihydroxyphe‐nylethylamine (DA, dopamine) and noradrenaline was examined in preparations of dog spleen and renal cortex following ultracentrifugation on a discontinuous sucrose gradient. In both tissues, only half the total tissue DA was localized to the soluble phase, and 30–50% was found in association with noradrenaline in the large vesicular fraction, suggesting that both catecholamines may be stored together and released by nerve stimulation. The vesicular fraction from renal cortex contained more DA than could be attributed to its presence in noradrenergic axons alone, supporting other evidence for the existence of dopaminergic renal nerves.
Effects of Opiates on High‐Affinity Ca2+,Mg2+‐ATPase in Brain Membrane Subfractions
Authors: Pillai N. P. , Ross D. H.
Published: October 2006

Abstract The effect of a single administration of morphine sulfate (15 mg/kg, s.c. or 30 mg/kg, i.p., 30 min) on Ca2+‐stimulated Mg2+‐dependent ATPase activity was investigated in synaptosomal plasma membranes (SPM) prepared from rat cortex. Morphine produced a significant decrease in Ca2+,Mg2+‐ATPase activity in synaptosomal fractions (SPM 1 + 2) known to contain a high density of opiate receptors and calmodulin‐dependent Ca2+,Mg2+‐ATPase. However, in another subpopulation (SPM 3) that contains fewer opiate receptors and less enzyme activity, no such decrease in the enzyme activity was observed after the opiate administration. The decrease in Ca2+,Mg2+‐ATPase activity seen in SPM 1 + 2 was specifically antagonized by the opiate antagonist naloxone hydrochloride (2 mg/kg, s.c.) when given 15 min before morphine administration. Mg2+‐ATP‐ase was not altered either by morphine or by a naloxone‐morphine combination. These findings give further evidence for the role of intracellular Ca2+ in mediating many of the acute effects of opiates.
Identification of Multiple Binding Sites for [3H]5‐Hydroxytryptamine in the Rat CNS
Authors: Blurton Pamela A. , Wood Martyn D.
Published: October 2006

Abstract: Recent studies indicate that there may be multiple subtypes of [3H]5‐hydroxytryptamine ([3H]5‐HT) binding sites. Mianserin and spiperone inhibited the specific binding of [3H]5‐HT (2–3 nM) to rat brain cortical membranes with shallow displacement curves. The displacement data for spiperone were best described by the presence of three independent binding sites, for which spiperone had high, medium, and low affinities. The displacement data for mianserin were best fitted by two independent, high‐ and low‐affinity sites. The inclusion of mianserin (250 nM) to inhibit [3H]5‐HT binding to the mianserin‐sensitive site selectively blocked one of the sites discriminated by spiperone. These results suggest the presence of three binding sites for [3H]]5‐HT. one blocked by low concentrations of spiperone (5‐HT1A), one blocked by low concentrations of mianserin (5‐HT1C), and one blocked only by high concentrations of both mianserin and spiperone (5‐HT1B). Regional differences in the relative densities of the three sites were observed. The hippocampus was rich in 5‐HT1A sites, whereas the striatum contained mainly 5‐HT1B and 5‐HT1C sites. Selective degeneration of 5‐HT‐containing nerve terminals induced by the neurotoxin 5,7‐dihydroxytryptamine increased binding to all three sites in the cerebral cortex. Binding of [3H]5‐HT to the three sites was differentially modulated by CaCl2 and guanylimidodiphosphate. The present data suggest the presence of three independent 5‐HT1 binding sites having different affinities for mianserin and spiperone and having different regional distributions.
Fatty Acid Composition of Endoneurium and Perineurium from Adult Rat Sciatic Nerve
Authors: Fressinaud C. , Rigaud M. , Vallat J. M.
Published: October 2006

Abstract: Reports on lipid composition of peripheral nervous system have generally been restricted to the saturated fatty acids of the endoneurium. In this work we attempt to determine the fatty acid composition of the different lipid classes in both endo‐ and perineurium from sciatic nerve microdissection on adult rats. Unsaturated fatty acids were found to make up around 60% of total fatty acids in samples of endoneurium and perineurium, with monounsaturated fatty acids forming 40–50% of total unsaturated fatty acid content. Although the same fatty acids were present in both tissues there was a striking difference in C 18:1 (n‐9) and C 18:2 (n‐6) ratio between endoneurium and perineurium, which is particularly rich in linoleic acid. The nonpolar perineurial lipids were found to be richest in linoleic acid. Phospholipids were present in the perineurium, and they contained high proportions of saturated and medium‐chain monounsaturated fatty acids.
Monoclonal Antibodies to and Immunoaffinity Purification of Choline Acetyltransferase from Bovine Brain
Authors: Ishida Isao , Ichikawa Tomoyuki , Deguchi Takeo
Published: October 2006

Abstract: Three hybridomas producing monoclonal antibodies to bovine brain choline acetyltransferase (ChAT) have been established by fusion of the spleen cells from a mouse immunized with purified enzyme with myeloma NS‐ 1 cells. All three clones produced IgGl antibodies that reacted with enzyme protein denatured with sodium dodecyl sulfate. By using one of the monoclonal antibodies, a rapid and efficient immunoaffinity purification procedure of bovine ChAT has been established. Immunoblot analysis and immunoaffinity purification indicated that bovine ChAT is a single 68‐kilodalton protein. The monoclonal antibodies will offer us a good tool to isolate the cDNA clones encoding ChAT.
Lipid Metabolism in Electroplax
Authors: Rotstein Nora P. , Arias Hugo R. , Aveldaño Marta I. , Barrantes Francisco J.
Published: October 2006

Abstract: The in vivo labeling of electrocyte lipids is followed after injection of radioactive glycerol and two fatty acids, oleate and arachidonate, into the electric organ of an elasmobranch (Discopyge tschudii). De novo synthesis of lipids and acyl‐exchange reactions are operative in the electrocyte. The three precursors are preferentially incorporated into phosphatidylcholine, phosphatidylinositol, and triacylglycerols. The highest specific activities are attained by triacylglycerols and polyphosphoinositides. Electrocyte stacks from electric organ show an efficient and continuous esterification of oleate and arachidonate into lipids after several hours of incubation. Except for an apparently more active labeling of triacylglycerols, which is attributed to the larger availability of free fatty acid precursors under the in vitro experimental conditions, the pattern of lipid labeling is similar to that attained in vivo. 32P‐labeled lipids are also steadily produced in electrocyte stacks (24 h of incubation with [32P]phosphate) using glucose as the sole exogenous source of energy. Polyphosphoinositides are the lipids preferentially labeled. The ability to sustain the labeling of lipids under in vitro conditions renders isolated electrocyte stacks an interesting model for future research on lipid involvement in cholinergic function.
Disproportional Expression of Proteolipid Protein and DM‐20 in the X‐Linked, Dysmyelinating Shaking Pup Mutant
Authors: Yanagisawa Katsuhiko , Moller Johanna R. , D. Duncan Ian , Quarles Richard H.
Published: October 2006

The shaking pup is an X‐linked canine mutant with a severe hypomyelination of the CNS. Proteolipid protein (PLP) and the related DM‐20 protein were examined in this mutant by densitometric scanning of Western blots stained with PLP antiserum. In the spinal cord of 4‐week‐old mutants, PLP was reduced to less than 1 % of the control level, which is a greater deficiency than was found for other myelin proteins. On Western blots of control spinal cord, PLP stained much more intensely than DM‐20. However, on Western blots of the mutant spinal cord, a component with the electrophoretic mobility of DM‐20 stained slightly more intensely with PLP antiserum than PLP itself. This component was shown to be DM‐20 by its lack of reactivity with an antiserum raised to a synthetic peptide corresponding to part of the PLP sequence that is missing in DM‐20. Thus PLP and DM‐20 are expressed in approximately equal and greatly reduced amounts in the mutant spinal cord. Although PLP or DM‐20 could not be detected in brain from the 4‐week‐old mutant, similar disproportional expression of these two proteins was demonstrated in both spinal cord and brain from a 10‐week‐old mutant pup. Immunostaining of tissue sections showed that the small amounts of PLP and/or DM‐20 synthesized in the mutant are present in the thin myelin sheaths. The results suggest that the shaking pup could have a primary defect in the PLP gene leading to a severe deficiency of PLP and DM‐20 as well as disproportional expression of these two proteins.
Characterization of K Opioid Receptors in Neurosecretosomes from Bovine Posterior Pituitary
Authors: Pesce Guido , Lang Michael A. , Russell James T. , Rodbard David , Gainer Harold
Published: October 2006

The binding properties of opioid receptors on isolated nerve terminals (neurosecretosomes) from bovine posterior pituitaries were characterized. Both [3H]etorphine and [3H]ethylketocyclazocine ([3H]EKC) showed high‐affinity binding with complex binding isotherms, consistent with the presence of multiple classes of binding sites. [D‐Ala2,D‐Leu5]enkephalin showed no specific binding and failed to displace [3H]etorphine at high concentrations, indicating the absence of μ, δ, or benzomorphan (K2) sites. Mathematical modelling of the data suggested the presence of three classes of binding sites. The first was of high affinity with Kd values of 0.9 and 2.0 nM for etorphine and EKC, respectively. The second class of sites appeared to bindetorphine with a KD of 150 nM, and EKC with extremely low affinity (unmeasurable binding). The third class of sites was characterized by KD values of 7 and 2 μM for etorphine and EKC, respectively. These results indicate that the nerve terminals of bovine posterior pituitary contain opioid binding sites of the K type. Futhermore, these binding sites appear heterogeneous, consisting of at least two and possibly more subtypes or states.