bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of Depolarization‐Dependent Release of Neurotransmitters by Adenosine: Cyclic AMP‐Dependent Enhancement of Release from PC12 Cells
Authors: Rabe Carolyn S. , McGee Richard
Published: October 2006

Abstract: We have used pheochromocytoma cells, clone PC12, as a model system for studying the effects of adenosine on neurosecretion. Exposure of the cells to adenosine or 2‐chloroadenosine caused immediate activation of adenylate cyclase, increases in cellular cyclic AMP content, and inhibition of SAM‐dependent phospholipid N‐methylation and protein carboxymethylation. However, the effects on methylation were only observed with concentrations of adenosine 100 times greater than those that elevated cyclic AMP. Exposure of the cells to adenosine and 2‐chloroadenosine did not alter the release of [3H]norepinephrine ([3H]NE) in the absence of depolarization. However, depolarization‐dependent release of [3H]NE was markedly elevated by short (1–20 min) pretreatments with adenosine or 2‐chloroadenosine. The enhancement of release was observed irrespective of the nature of the depolarizing stimulus (elevated K+, carbamylcholine, or veratridine). Release of [3H]acetylcholine in response to elevated K+ also was increased by adenosine pretreatment. These effects of adenosine and 2‐chloroadenosine on neurotransmitter release closely paralleled elevation of cellular cyclic AMP but not inhibition of methylation. Taken together, the results show that adenosine, probably acting through adenosine receptors coupled to stimulation of adenylate cyclase, is able to modulate the neurosecretory process in PC12 cells. Furthermore, the enhancement of release occurred even though the extent of depolarization (measured as 86Rb+ flux through the acetylcholine receptor channel) and the amount of 45Ca2+ which entered upon depolarization were unchanged. Therefore, the enhancement of release produced by elevated cyclic AMP appeared to reflect increased efficiency of the stimulus‐secretion coupling process.
Effect of Sound Stimulation at Several Levels on Concentrations of Primary Amines, Including Neurotransmitter Candidates, in Perilymph of the Guinea Pig Inner Ear
Authors: Drescher Marian J. , Drescher Dennis G. , Medina Jesus E.
Published: October 2006

Abstract: Perilymph, which bathes the sensory cells of the cochlea, was collected from guinea pigs exposed to noise and analyzed via two cation‐exchange HPLC procedures with fluorescence detection, resolving 51 and 81 primary‐amine compounds, respectively, at a sensitivity limit of 0.1 pmol relative to leucine. During a first period, each animal was either exposed to noise at 80, 90, or 115 decibels sound‐pressure level or maintained in silence (controls), and during a second period, the same animal was maintained in silence. Perilymph was collected during both periods, and perilymphatic components were compared, within animals and across animals, for several levels of sound stimulation. A 7‐aminobutyric acid‐like component was elevated in the first period in proportion to stimulus intensity by the various methods of comparison, suggesting an auditory‐neurotransmitter role for this component. Aspartic acid was elevated in the second period, 2–3.5 h after onset of sound stimulation, compatible with the release of aspartic acid from central auditory synapses. In addition, a methionine‐enkephalin‐like component, distinct from leucine‐enkephalin, was detected in perilymph from control animals and was elevated in response to noise at 115 decibels. Regression coefficients, determined for the relation between sound intensity and first‐period concentrations or the difference between first and second‐period concentrations, indicated zero linear regression at p = 0.05 for glutamic acid, aspartic acid, glycine, taurine, and 39 other perilymphatic components, consistent with the hypothesis that these compounds are unlikely to be peripheral auditory neurotransmitters.
Influence of the Ionic Environment on the Conformation of Aspartic Acid and Possible Relevance to Its Neurotransmitter Action
Authors: Esposito Gennaro , Motta Andrea , Temussi Piero A.
Published: October 2006

The conformational changes of aspartic acid, a possible neurotransmitter, induced by increasing ionic strength were investigated by means of NMR spectros‐copy. The effects of Li, Na, K, and Cs chlorides in basic and neutral conditions were characterized. The effects of the presence of both Ca2+ and Na+ or K+ were also examined. The results always showed a relative stabilization of the two conformers with gauche carboxylates at the expense of the anticonformer. The significant quantitative difference found between Na+ and K+ in basic solutions, as well as between calcium‐free and calcium‐containing solutions, may bear some relevance for the role of aspartate as a CNS neurotransmitter.
Characterization and Location of Met5‐Enkephalin‐Arg6‐Phe7 Stored in Various Rat Brain Regions
Authors: Yang H.Y. T. , Panula P. , Tang J. , Costa E.
Published: October 2006

A specific antiserum against met5‐enkepha‐lin‐arg6‐phe7 was raised and used to study the distribution and characterization of met5‐enkephalin‐arg6‐phe7‐like immunoreactive material in rat brains by radioimmunoassay and immunohistochemical procedures. The antiserum appears to be directed to the COOH‐terminus of the peptide, as it fails to cross‐react with met5‐enkeph‐alin, met3‐enkephalin‐arg6, met5‐enkephalin‐arg6‐arg7, met6‐enkephalin‐lys6, and leu‐enkephalin. However, it cross‐reacts with phe‐met‐arg‐phe by about 10% and with phe‐met‐arg‐phe‐NH2 to an insignificant degree. The highest content of met5‐enkephalin‐arg6‐phe7 was found in the striatum, which contains a dense network of immunoreactive varicose fibers and terminals, as well as immunoreac tive cell bodies. The met5‐enkephalin‐arg6‐phe7 in striatum can be released in a Ca2+‐dependent manner by a depolarizing concentration of KC1, raising the possibility of a neu‐roregulatory role for met5‐enkephalin‐arg6‐phe7. Characterization of the immunoreactive material by gel filtration and high pressure liquid chromatography revealed the presence of multiple forms of immunoreactive material in some brain regions.
Characterization of the Ca2+‐ and Mg2+‐Dependent ATPases in Electrophorus Electroplax Microsomes
Authors: Amende L. M. , Chock S. P. , Albers R. W.
Published: October 2006

Electrophorus electroplax microsomes were examined for Ca2+‐ and Mg2+‐dependent ATPase activity. In addition to the previously reported low‐affinity ATPase, a high‐affinity (Ca2+,Mg2+)‐ATPase was found. At low ATP and Mg2+ concentrations (200 μM or less), the high‐affinity (Ca2+,Mg2+)‐ATPase exhibits an activity of 18 nmol Pi, mg‐1min‐1 with 0.58 μM Ca2+. At higher ATP concentrations (3 mM), the low‐affinity Ca2+‐ATPase predominates, with an activity of 28 nmol Pi mg‐1min‐1 with 1 mM Ca2+. In addition, Mg2+ can also activate the low‐affinity ATPase (18 nmol Pi mg‐1min‐1). The high‐affinity ATPase hydrolyzes ATP at a greater rate than it does GTP, ITP, or UTP and is insensitive to ouabain, oligomycin, or dicyclohexylcarbodiimide inhibition. The high‐affinity enzyme is inhibited by vanadate, tri‐fluoperazine, and N‐ethylmaleimide. Added calmodulin does not significantly stimulate enzyme activity; rinsing the microsomes with EGTA does not confer calmodulin sensitivity. Thus the high‐affinity ATPase from electroplax microsomes is similar to the (Ca2+,Mg2+)‐ATPase reported to be associated with Ca2+ transport, based on its affinity for calcium and its response to inhibitors. The low‐affinity enzyme hydrolyzes all tested nucleoside triphosphates, as well as diphosphates, but not AMP. Vanadate and N‐ethylmaleimide do not inhibit the low‐affinity enzymes. The low‐affinity enzyme reflects a nonspecific nucleoside triphosphatase, probably an ectoenzyme.
Synaptosomal and Brain Mitochondrial Lipids in Hibernating and Cold‐Acclimated Golden Hamsters
Authors: Demediuk Paul , Moscatelli Ezio A.
Published: October 2006

Synaptosomes and mitochondria were isolated from the brains of warm‐adapted, hibernating, and cold‐acclimated golden hamsters (Mesocricetus auratus). Lipid extracts of these subcellular fractions were prepared and assayed for plasmenylethanolamin (ethanolamine plasmalogen) and cholesterol levels. The ganglioside composition of synaptosomes was also determined. Samples from the hibernating animals showed characteristic changes in lipid composition. These changes include decreases in plasmenylethanolamine levels and a shift in the ganglioside composition toward a higher percentage of the more polar gangliosides. Those animals which were exposed to cold and did not hibernate (cold‐acclimated) showed no such changes. Fatty acid analyses of synaptosomal and mitochondrial ethanolamine glycerophospholipids demonstrated a similar trend. Samples from hibernators showed decreases in 16:0, 18:0, and 22:6 (n‐3), and increases in 16:1, 18:1, and 20:4 (n‐6) fatty acids. No changes were detectable in samples from cold‐acclimated animals, indicating that hibernating and cold‐acclimated hamsters represent chemically distinct populations.
Molecular Forms of Acetylcholinesterase: Regulation in a Testosterone‐Sensitive Nerve‐Muscle Axis
Authors: Bleisch William V. , Luine Victoria N. , McEwen Bruce S.
Published: October 2006

We measured the distribution of molecular forms of acetylcholinesterase (AChE) in muscles of a song bird, the zebra finch, and found a pattern similar to those reported in other vertebrates. As in other species, the most rapidly sedimenting form of the enzyme decreases to barely detectable levels following denervation. In the muscles of the syrinx, castration causes a large decrease in AChE activity, but has little or no effect on the relative abundance of AChE forms. This suggests that the number of AChE catalytic sites is changing without affecting the distribution of catalytic sites among the molecular forms. This is in marked contrast with the effect of denervation in the syrinx, which causes changes in the distribution of activity, as well as in total activity.
Light Enhances the Turnover of Phosphatidylinositol in Rat Retinas
Authors: Schmidt Susan Y.
Published: October 2006

Light stimulation of isolated rat retinas is shown to enhance the turnover of phosphatidylinositol (PI) as demonstrated by a light‐dependent increase in [3H]inositol incorporation and concurrent hydrolysis of existing PI. Studies with rat retinas incubated with [3H]inositol and then microdissected at the level of the outer plexiform layer into photoreceptor cell and inner retina layers indicated that the light‐enhanced incorporation of [3H]inositol was associated with the photoreceptor cell layer. The rate of PI hydrolysis in retinas prelabeled in vivo with [3H]inositol was higher in light than in dark incubations and was higher in the photoreceptor cell layer than within the inner retina. Within the photoreceptor cell layer, PI turnover involved 2%/min of the total PI contentin dark and 6–8%/min in light. In contrast to what has been reported for stimulus‐enhanced turnover of PI in some tissues, this light‐enhanced turnover of PI in the retina was not associated with detectable reductions in PI content. Parallel studies of sodium (22Na) uptake demonstrated that the photoreceptor cells remained functional during these incubations as they retained the capacity to restrict the entry of 22Na in light but not in dark.
Uptake and Release of D‐Aspartate in the Guinea Pig Cochlear Nucleus
Authors: Potashner S. J.
Published: October 2006

Abstract: This study attempted to determine if l‐glutamate (L‐Glu) and/or l‐aspartate (L‐Asp) might be the transmitters of neurons that provide synaptic endings to the cochlear nucleus of the medulla. The uptake and release of D‐[3H]aspartate (D‐Asp), a putative marker for l‐Glu and l‐Asp, were measured in the guinea pig cochlear nucleus before and after destruction of the cochlear afferents by cochlear ablation. The cochlear nucleus was dissected into the anteroventral (AVCN), posteroventral (PVCN), and dorsal (DCN) cochlear nuclei. Subdivisions from unlesioned animals took up D‐Asp, achieving concentrations in the tissues that were 13–20 times that in the medium. Subsequently, electrical stimulation evoked a Ca2+‐dependent release of part of the D‐Asp from each subdivision. Disarticulation of the middle ear ossicles, which attenuates acoustic stimulation, produced a modest inhibition of D‐Asp release in each subdivision, but did not alter the uptake of D‐Asp. Cochlear ablation strongly depressed both the uptake and the release of D‐Asp in each subdivision, presumably as a result of destruction of the cochlear nerve endings in the cochlear nucleus. Nevertheless, after lesions, there was a preservation of the uptake and release of D‐Asp in the DCN relative to the AVCN and PVCN. These residual activities in the DCN may be mediated by the axonal endings of the granule cells of the cochlear nucleus. The present findings support the hypothesis that the granule cells of the cochlear nucleus, as well as the cochlear nerve fibers, use l‐Glu and/or l‐Asp as transmitters.
Occurrence of 5′‐Deoxy‐5′‐Methylthioadenosine Phosphorylase in the Mammalian CNS: Distribution and Kinetic Studies on the Rat Brain Enzyme
Authors: Abbruzzese Alberto , Pietra Gennaro Delia , Porta Raffaele
Published: October 2006

Abstract: 5′‐Deoxy‐5′‐methylthioadenosine (MTA) phosphorylase catalyzes the cleavage of MTA, a secondary product of polyamine biosynthesis, to 5‐methylthiori‐bose‐1‐phosphate and adenine. The occurrence and the general properties of the enzyme were studied in mammalian brain with the following results. (1) Cerebral tissues contained levels of MTA phosphorylase that were comparable to those occurring in other mammalian tissues. (2) Interspecies differences in the enzyme distribution were quite limited, with the highest specific activity values observed in pig brain. Moreover, the enzyme seemed to be generally more concentrated in the cerebellar fractions. (3) Rat brain MTA phosphorylase was highly localized in the cellular soluble fraction. In the first days of rat life, its specific activity in the whole brain was observed to decline significantly from a value of 17.6 units/mg at 1–5 days of age to 13.7 units/mg at 6–10 days of age, remaining then fairly constant up to maturity. (4) Kinetic studies performed with the soluble enzyme extracted from rat brain showed: a pH optimum of 7.4; a Km value for MTA of about 10 μM; an inhibitory effect of the MTA analog 5′‐deoxy‐5′‐isobutylthioadenosine; and a remarkable resistance of the enzyme to heat treatment.
Accumulation of Phosphatidic Acid in Microsomes from Propranolol‐Treated Retinas During Short‐Term Incubations
Authors: Giusto Norma M. , Ilincheta de Boschero Monica G. , Bazan Nicolas G.
Published: October 2006

Abstract: The pool size and synthesis of phosphatidic acid derived from [2‐3H]glycerol were studied in bovine whole retinas and subcellular fractions. Microsomal preparations from retinas incubated with [2‐3H]glycerol displayed the highest percentage labeling of phosphatidic acid at 5 min of incubation; labeling decreased rapidly thereafter. In drug‐treated retinas,0.5 mM propranolol increased the endogenous content of phosphatidic acid and stimulated [2‐3H]glycerol labeling in whole retina and microsomal and postmicrosomal supernatant fractions. This effect was observed during short‐term incubations and was reversible. In pulse‐chase experiments, 60 min of reincubation greatly reduced the labeling effect, although propranolol still enhanced phosphatidic acid labeling. At the same time, endogenous phosphatidic acid accumulated and reincubation without propranolol reversed the effect. During accumulation, the amount of palmitate increased and that of oleate decreased, whereas the relatively high level of docosahexaenoate in phosphatidic acid remained unchanged. It was concluded that this propranolol‐induced effect is due to cationic amphiphilic drug activity in the endoplasmic reticulum that results in a partial inhibition of phosphatidic acid degradation and a stimulation of its de novo synthesis. Hence, net synthesis of phosphatidic acid can be assessed in the retina during short‐term incubation with propranolol.
Irreversible Binding of [3H]Flunitrazepam to Different Proteins in Various Brain Regions
Authors: Sieghart W. , Drexler G.
Published: October 2006

Abstract: Irreversible photolabeling by [3H]flunitraze‐pam of four proteins with apparent molecular weights 51,000 (P51), 53,000 (P53), 55,000 (P55), and 59,000 (P59) was investigated in various rat brain regions by SDS‐polyac‐rylamide gel electrophoresis, fluorography, and quantitative determination of radioactivity bound to proteins. On maximal labeling of these proteins, only 15–25% of [3H]flunitrazepam reversibly bound to membranes becomes irreversibly attached to proteins. Results presented indicate that for every [3H]flunitrazepam molecule irreversibly bound to membranes, three molecules dissociate from reversible benzodiazepine binding sites. This seems to indicate that these proteins are either closely associated or identical with reversible benzodiazepine binding sites, and supports the hypothesis that four benzodiazepine binding sites are associated with one benzodiazepine receptor. When irreversible labeling profiles of proteins P51, P53, P55, and P59 were compared in different brain regions, it was found that labeling of individual proteins varied independently, supporting previous evidence that these proteins are associated with distinct benzodiazepine receptors.
Solubilization of Active Brain α1‐Adrenoceptors by a Zwitterionic Detergent
Authors: Guicheney Pascale , Rappaport Alain , Marcel Dominique
Published: October 2006

Abstract: Solubilization of rat brain α1‐adrenoceptors was performed by treatment with 6 mM CHAPS (3–[(3‐cholamidopropyl)dimethylammonio] ‐ 1 ‐ propanesulfon‐ate). The α1‐adrenoceptor antagonist [3H]prazosin was shown to bind reversibly and specifically to the soluble extract obtained after centrifugation at 150,000 ±g for 1 h. Separation of the soluble [3H]prazosin‐bound complexes was performed by the polyethylene glycol precipitation technique followed by filtration. A Scatchard plot of the concentration‐dependent binding curve showed only one class of binding sites, with a high affinity for [3H]prazosin. Affinity of the solubilized receptors for the ligand increased as the CHAPS concentration in the assay medium decreased; the number of binding sites remained unchanged (=70 fmol/mg protein). This corresponds to a 30% recovery of original membrane sites. The solubilized receptors presented the same characteristics of specificity and stereospecificity as membrane α1‐adrenoceptors. Moreover, 150 mM NaCl was found to modulate the affinity of epinephrine for the [3H]prazosin‐bound soluble complex, as previously described for membrane preparations. Thus, CHAPS appears to be a suitable detergent for solubilizing rat brain α1‐adrenoceptors and preserving their functional activities.
Neopterin Retards Loss of Serotonin N‐Acetyltransferase Activity from Cultured Chick Pineal Glands
Authors: Wainwright S. D. , Wainwright Lillian K.
Published: October 2006

Abstract: d‐Neopterin at 10 μM delayed start of the decline of serotonin N‐acetyltransferase (NAT) activity from the peak level in the cycle exhibited by chick pineal glands cultured under standard conditions in the dark. A less marked retardation of decline of NAT activity was found with glands cultured under diurnal illumination or those exposed prematurely to light. There were no significant effects of neopterin on the increases of NAT activity or peak levels of activity developed. The pteridine also retarded loss of NAT activity from the peak level developed in the dark when the time of explanting into culture was later in the (solar) day, but not when it was earlier. Neopterin had no effect on the cycle in cyclic GMP content of cultured chick pineal glands.
Characterization and Biosynthesis of Cyclic‐AMP‐Binding Proteins in the Rat Central Nervous System
Authors: Strocchi Paola , Sapirstein Victor S. , Rubin Charles S. , Gilbert Jeffrey M.
Published: October 2006

Abstract: Cyclic‐AMP‐binding proteins in membrane and soluble fractions from rat forebrain were compared; membrane fractions included smooth and rough microsomes and a plasma membrane fraction enriched in synaptic membranes. Protein fractions were treated with 8‐azido‐[32P]cyclic AMP and ultraviolet irradiation to covalently tag cyclic‐AMP‐binding proteins. Labeled proteins were then analyzed by two‐dimensional gel electrophoresis (2DGE) and fluorography. The soluble CNS proteins contained two major cyclic‐AMP‐binding species at 48K (48K 5.5 and 48K 5.45), differing slightly in their isoelectric points. Another protein was seen at 54K (54K 5.3) adjacent to the β‐tubulin subunits in the 2D electrophoretogram. The analysis of the smooth microsome and plasma membrane fractions differed from the soluble fraction in that there were two cyclic‐AMP‐binding proteins adjacent to the β‐tubulin region (54K 5.3 and 52K 5.3) differing slightly in apparent molecular weight. The membrane fractions also contained a cyclic‐AMP‐binding protein at 54K 5.8. The 52K 5.3 and 54K 5.8 species were unique to the membrane fractions. The rough microsomes did not contain detectable amounts of cyclic‐AMP‐binding proteins. Free polysomes were isolated from brain tissue, and translation products were analyzed by cyclic AMP affinity chromatography and immunopurification with antibodies to the brain specific type II regulatory subunit. The translation products that were found to bind cyclic AMP Sepharose are as follows: 48K 5.5, 48K 5.45, 52K 5.3, and 54K 5.8. These species comigrated with proteins that were photoaffmity‐labeled in cytosol and membrane fractions. A translation product at 54K 5.3 was not detected, and, therefore, the possibility exists that this protein may result from posttranslational modification of another protein. The translation product at 52K 5.3 was precipitated with antibodies against the brain specific type II regulatory subunit of cyclic‐AMP‐dependent protein kinase.
Analysis of the Acid Phosphatases of Rat Sensory Gangli by Isoelectric Focussing on Polyacrylamide Gels
Authors: Bahsoon J. D. , Hyde Sandra , Keen P.
Published: October 2006

Abstract: The acid phosphatases of rat spinal and trigeminal ganglia have been separated by isoelectric focussing on polyacrylamide gels into two main classes with pI's of 7.1 and 5.4. A low‐pI acid phosphatase is also present in the mouse but not in the guinea pig. Evidence is presented that in the rat the pI 5.4 enzyme represents the nonlysosomal acid phosphatase that has been identified histochemically in one population of sensory neurones. This pI 5.4 acid phosphatase is partly membrane‐bound and is selectively depleted by capsaicin administration. In lumbar dorsal root ganglia the enzyme is selectively depleted by sectioning the sciatic nerve and undergoes rapid axonal transport, a greater amount being transported peripherally than centrally. The results are discussed with reference to the possible function of this nonlysosomal acid phosphatase.
Low Buoyant Density Proteoglycans from Saline and Dissociative Extracts of Embryonic Chicken Retinas
Authors: Morris John E. , Ting Yuan‐Pin , Birkholz‐Lambrecht Anne
Published: October 2006

Abstract: Retinas were labeled in culture with [3H]glucosamine or [3H]leucine and [35S]sulfate and extracted sequentially with physiologically balanced saline and 4 M guanidine HCl. They were dialyzed into associative conditions (0.5 M NaCl) and chromatographed on agarose columns. Under these conditions, some of the proteoglycans were associated in massive complexes that showed low buoyant densities when centrifuged in CsCl density gradients under dissociative conditions (4 M guanidine HCl). Much of the label in these complexes was in molecules other than proteoglycans. Most of the proteoglycans, however, were included on the agarose columns, where they appeared to be constitutionally of low buoyant density. They resisted attempts to separate potential low buoyant density contaminants from the major proteoglycans by direct CsCl density gradient centrifugation or by the fractionation of saline or 8 M urea extracts on diethylaminoethyl‐Sephacel. The diethylaminoethyl‐Sephacel fractions were either subjected to CsCl density gradient centrifugation or were chromatographed on Sephacryl S‐300, in both cases before and after alkaline cleavage, to confirm the presence of typical O‐linked glycosaminoglycans. The medium and balanced salt extracts were enriched in chondroitin sulfate and other sul‐fated macromolecules, possibly highly sulfated oligosaccharides, that resisted digestion by chondroitinase ABC but were electrophoretically less mobile than heparan sulfate. Guanidine HCl or urea extracts of the residues were mixtures of high and low density proteoglycans that were enriched in heparan sulfate.
Choline Acetyltransferase and Cholinesterases in the Developing Xenopus Retina
Authors: Ma PoKay M. , Grant Philip
Published: October 2006

Abstract: To understand the developmental regulation of acetylcholine (ACh) synthesis in the Xenopus retina, the properties of choline acetyltransferase (CAT) and cholin‐esterase (ChE), as well as histochemical localization of ChE in the retina, were studied during development. CAT activity first became detectable in the developing eyecup at stages 35/36. This was followed by a rapid, 50‐fold rise in specific activity between stages 35/36 and 44. Since this rapid rise coincided with an almost identical increase in total ACh synthesis in whole retinae found in previous studies, it is suggested that this increase was sufficient to account for the rapid increase in total ACh synthesis. Moreover, it also correlated with increased rates of synaptogenesis in both the inner and the outer plexiform layers. Total ChE was resolved into specific and nonspecific ChE by the use of tetraisopropylpyrophosphor‐amide. Total ChE activities first became detectable at stages 35/36. Specific ChE [acetylcholinesterase (AChE)] increased from 50% at stage 39 to 95% of total ChE activities at stage 66. Again, the most rapid increase in both total ChE and AChE activities occurred between stages 35/36 and 44. Histochemical studies showed that AChE was localized predominantly in the two plexiform layers, with the inner plexiform layer more heavily stained at all stages. Moreover, a stratified staining pattern, clearly discerned in the inner plexiform layer, also correlated with synaptogenesis during this early period of retinal development.
Extraction and Partial Characterization of Enkephalin‐Like Peptides from Splanchnic Nerve
Authors: Hexum Terry D. , Russett Linda R. , Barren Barbara A.
Published: October 2006

Abstract: A method is described for the extraction of enkephalin‐like peptides from peripheral nerve using chloroform and acidic methanol to facilitate a differential extraction of peptides and lipid. Porcine splanchnic nerve contains enkephalin‐like peptides in low amounts compared to porcine adrenal medulla and striatum. Gel filtration chromatography reveals the presence of enkephalinlike peptides in both processed and cryptic forms. This is the first reported isolation and partial characterization of these peptides in splanchnic nerve. The presence of these peptides in this nerve provides support for the contention that the splanchnic nerve can modulate catecholamine release from the adrenal medulla through an effect on opiate receptors located on chromaffin cells.
Elevation of Intracellular Cyclic AMP and Stimulation of Adenylate Cyclase Activity by Vasoactive Intestinal Peptide and Glucaeon in the Retinal Pigment Epithelium
Authors: Koh Shav‐Whey M. , Chader Gerald J.
Published: October 2006

Both vasoactive intestinal peptide (VIP) and glucagon rapidly elevated cyclic AMP levels in embryonic chick retinal pigment epithelium (RPE), in culture as well as in freshly dissected tissue. In cultured cells, the half‐maximal activities of VIP and glucagon were 5 × 10‐8M and 3 × 10‐8M, respectively. After 3 min of reaction, VIP elevated intracellular cyclic AMP by 100‐fold; elevation with glucagon was up to 10‐fold. Both neuropeptides stimulated adenylate cyclase activity in RPE membranes. Glucagon showed a half‐maximal activity of 1 × 10‐8M. VIP remained more effective than glucagon in stimulating adenylate cyclase activity, but the doseresponse curve was shifted to a higher concentration range when compared to that of the VIP‐elevated intracellular cyclic AMP.
Reciprocal Regulation of Ornithine Decarboxylase and Choline Kinase Activities by Their Respective Reaction Products in the Developing Rat Cerebellar Cortex
Authors: Gilad Gad M. , Gilad Varda H.
Published: October 2006

Changes in the activity of choline kinase were measured in the cerebellum during development. Early transient increase was found in the enzyme activity just prior to and during birth. This period of increase did not coincide with the periods of transient elevation in ornithine decarboxylase and choline acetyltransferase previously observed in the developing cerebellum. The effects of the naturally occurring polyamines (putrescine, spermidine, and spermine) on choline kinase and choline acetyltransferase activities, and of phosphorylcholine (the product of the reaction catalyzed by choline kinase) on ornithine decarboxylase and choline acetyltransferase activities, were also examined. Choline acetyltransferase activity was not influenced by either polyamines or phosphorylcholine. However, choline kinase activity from 7‐day‐old, but not from adult, cerebellum was increased 25% in the presence of 4 mM spermine. In contrast, low spermidine concentrations (<2 mM) inhibited choline kinase activity selectively in 7‐day‐old cerebellum. Ornithine decarboxylase activity from 7‐day‐old cerebellum was inhibited in a concentration‐dependent manner by phosphorylcholine. The present data together with other previous reports suggest that: (a) polyamines may play a role in choline utilization during development via their regulation of choline kinase activity, on the one hand, and of acetylcholinesterase activity on the other; and (b) during development, a reciprocal regulation of choline kinase and ornithine decarboxylase activities by their respective reaction products may exist, whereby choline kinase activity is regulated in a complex manner by polyamines and, in turn, ornithine decarboxylase is inhibited by phosphorylcholine.
Degradation of NAD by Synaptosomes and Its Inhibition by Nicotinamide Mononucleotide: Implications for the Role of NAD as a Synaptic Modulator
Authors: Snell C. R. , Snell P. H. , Richards C. D.
Published: October 2006

We have found NAD to be rapidly degraded by extracellular enzymes present on intact rat brain synaptosomes. The enzyme involved had the specificity of an NADase cleaving the molecule at the nicotinamide‐glycoside linkage and was inhibited by nicotinamide mononucleotide (NMN). This inhibitor did not displace specific binding of NAD to rat brain membranes or affect electrical activity in the guinea pig hippocampus. Therefore, inclusion of NMN in binding assays allowed unambiguous demonstration of two specific NAD binding sites on rat brain synaptosomal membranes (KD1, 82 nM, KD2, 1.98 μM). The depressant action of NAD on the evoked synaptic activity of the guinea pig hippocampus was not blocked after inhibition of NAD degradation with NMN. The physiological implications of these results for the function of NAD as a neurotransmitter or neuro‐modulator in the CNS are discussed.
GABA Induces Functionally Active Low‐Affinity GABA Receptors on Cultured Cerebellar Granule Cells
Authors: Meier Eddi , Drejer Jørgen , Schousboe Arne
Published: October 2006

The effect of γ‐aminobutyric acid (GABA) and its agonists muscimol and 4,5,6,7‐tetrahydroisoxazolo[5‐4‐c]pyridin‐3‐ol (THIP) on the development of GABA receptors on cerebellar granule cells was studied by cultivation of the cells in media containing these substances. It was found that the presence of 50 μM GABA in the culture media led to the induction of low‐affinity GABA receptors (KD 546 ± 117 nM) in addition to the high‐affinity receptors (KD 7 ± 0.5 nM) which were present regardless of the presence of GABA in the culture media. The functional activity of the GABA receptors was tested by investigating the ability of GABA to modulate evoked glutamate release from the cells. It was found that GABA could inhibit evoked glutamate release (ED50 10 ± 3 (μM) only when the cells had been cultured in the presence of 50 νM GABA, 50 μM muscimol, or 150 μM THIP, i.e., under conditions where low‐affinity GABA receptors were present on the cells. This inhibitory effect of GABA could be blocked by 120 μM bicuculline and mimicked by 50 μM muscimol or 150 μM THIP whereas 150 μM (‐)‐baclofen had no effect. It is concluded that GABA acting extracellularly induces formation of low‐affinity receptors on cerebellar granule cells and that these receptors are necessary for mediating an inhibitory effect of GABA on evoked glutamate release. The pharmacological properties of these GABA receptors indicate that they belong to the so‐called GABAA receptors.
Interaction of Neuropeptides and Cultured Glial (Müller) Cells of the Chick Retina: Elevation of Intracellular Cyclic AMP by Vasoactive Intestinal Peptide and Glucagon
Authors: Koh Shay‐Whey M. , Kyritsis A. , Chader Gerald J.
Published: October 2006

Abstract: Vasoactive intestinal peptide (VIP) and, to a lesser extent, glucagon were found to increase intraclular cyclic AMP rapidly in cultured glial (Müller) cells of the chick embryo retina. Although VIP elicited higher cyclic AMP accumulation than glucagon at each concentration tested, the half‐maximal concentrations were similar, i.e., 6 × 10−8M for VIP and 8 × 10−8M for glucagon. Secretin had a minimal effect on cyclic AMP accumulation even at a very high (5 × 10−6M) concentration. Several other peptide and nonpeptide putative agonists also had little effect on cyclic AMP accumulation. The cultured Müller cell may thus be a useful model for examining VIP and glucagon effects on glial elements of the CNS.
Measurement of Acetylcholine Turnover Rate in Brain: An Adjunct to a Simple HPLC Method for Choline and Acetylcholine
Authors: Potter P. E. , Hadjiconstantinou M. , Meek J. L. , Neff N. H.
Published: October 2006

Abstract: An existing method for measuring acetylcholine (ACh) and choline (Ch) is shown to be useful formeasuring the turnover rate of ACh in mouse brain. Methl‐[3H]Ch is injected into mice. They are killed atdifferent times by microwave irradiation and Ch and AChextracted and separated by reverse‐phase HPLC. Ch andACh are converted to hydrogen peroxide by a post‐column enzyme reaction. Hydrogen peroxide, which isdirectly related to the tissue content of Ch or ACh, isdetermined electrochemically. The fractions that corre‐spond to the detector response for Ch and ACh are col‐lected for the measurement of radioactivity. In this wayspecific radioactivities of endogenous Ch and ACh areestimated in the same sample. We used the specific ra‐dioactivity values determined by this procedure to esti‐mate the turnover of ACh for striatum, cerebral cortex, and hippocampus of the mouse.
Postmortem Changes in Catecholamines, Indoleamines, and Their Metabolites in Rat Brain Regions: Prevention with 10‐kW Microwave Irradiation
Authors: Ikarashi Yasushi , Sasahara Tomiya , Maruyama Yuji
Published: October 2006

Abstract: Postmortem changes in catecholamines, indoleamines, and their metabolites in rat brain regions following decapitation were determined by LCEC. In the three regions studied neurotransmitter levels declined after decapitation, whereas the metabolite levels increased. Microwave irradiation at 10 kW rapidly inactivated brain enzymes and thus prevented the postmortem changes.
N‐Methylaspartate‐Evoked Liberation of Taurine and Phosphoethanolamine In Vivo: Site of Release
Authors: Lehmann Anders , Lazarewicz Jerzy W. , Zeise Marc
Published: October 2006

Abstract: The effect of N‐methyl‐d,l‐aspartic acid (NMA) on extracellular amino acids was studied in the rabbit hippocampus with the brain dialysis technique. Administration of 0.5 or 5 mM NMA caused a concentration‐dependent liberation of taurine and phosphoethanolamine (PEA). Taurine increased by 1,200% and PEA by 2,400% during perfusion with 5 mM NMA whereas most other amino acids rose by 20–100%. The effect of NMA appeared to be receptor‐mediated, as coperfusion with D‐2‐amino‐5‐phosphonovaleric acid curtailed the NMA response by some 90%. The NMA‐stimulated release of taurine and PEA was suppressed when Ca2+ was omitted and further inhibited when Co2+ was included in the perfusion medium. The effect of NMA was mimicked by the endogenous NMA agonist quinolinic acid and the partial NMA agonist d,l‐cis‐2,3‐piperidine dicarboxylic acid. Although the NMA‐evoked release of taurine and PEA was Ca2+‐dependent in vivo, NMA had no effect on Ca2+ accumulation in hippocampal synaptosomes. The previously reported NMA‐induced activation of dendritic Ca2+ spikes and the lack of effect on synaptosomal Ca2+ uptake suggest that taurine and PEA are released from sites other than nerve terminals, possibly from dendrosomatic sites. This notion was strengthened by the absence of an effect of NMA on the efflux of radiolabelled taurine from hippocampal synaptosomes. In contrast, high K+ stimulated synaptosomal uptake of Ca2+ and release of taurine.
Catabolism of myo‐Inositol to Precursors Utilized for De Novo Glycerolipid Biosynthesis
Authors: Fliesler Steven J. , Kelleher Paula A. , Anderson Robert E.
Published: October 2006

Systemic injection of [2‐3H]myo‐inositol into frogs resulted in the incorporation of more than half of the label into glycerolipid classes other than phosphoinositides in retinal rod outer segment membranes. Following methanolysis and differential extraction of isolated lipid classes, radioactivity was recovered primarily in the aqueous phase. After phospholipase C hydrolysis of the total membrane lipids, 97% of the radioactivity was extractable with organic solvents, and 70% of the label in lipids was in 1,2‐diglycerides. These results indicate that the label was incorporated primarily into the glyceryl moiety of the membrane glycerolipids. Intraocular injection of frog eyes or in vitro incubation of frog retinas with [2‐3H]myo‐inositol resulted in the incorporation of radioactivity almost exclusively into phosphoinositides in rod outer segment membranes. Incubation of retinas with [U‐14C]glucuronic acid did not result in the formation of labeled retinal lipids. These results suggest that myo‐inositol can be catabolized systemically to precursors utilized for glycerolipid biosynthesis in the retina.
Release of Purines, Noradrenaline, and GABA from Rat Hippocampal Slices by Field Stimulation
Authors: Jonzon Bror , Fredholm Bertil B.
Published: October 2006

Labelled adenine, noradrenaline (NA), and γ‐aminobutyric acid (GABA) were taken up by the transversely cut hippocampal slice. [3H]NA and [14C]GABA were retained as such, [3H]‐ (or [14C]‐) adenine mainly as adenine nucleotides. There was a spontaneous overflow of all three types of compounds ranging from 0.1 (GABA) to 0.21 (NA) %/min. The rate of [3H]NA overflow increased rapidly during electrical field stimulation. The release rate was well maintained over a 15‐min period. The rate of [14C]GABA release also increased rapidly but it was not maintained over a 15‐min period even if uptake and/or metabolism was inhibited by nipecotic acid (1 mM) and aminooxyacetic acid (AOAA, 0.1 mM). The bulk of the purines was released after the stimulation period. For all compounds the amounts released were frequency‐ and calcium‐dependent. At a frequency of 3 Hz a 10 V stimulation was sufficient to cause a maximal [3H]NA release and 20 V to cause maximal [14C]GABA release, but 14C‐purine release was increased further by increasing the voltage to 40 V. The evoked purine release was inhibited by a nucleoside uptake inhibitor (dipyridamole). On stimulation of [3H]NA‐labelled slices the released radioactivity was composed of >95% unchanged NA. The specific activities of NA in the slice and in the superfusate were practically identical. In [3H]adenine‐labelled slices the released radioactivity was composed of adenosine, inosine, and hypoxanthine, but the activity in the slice of ATP, ADP, and AMP. The specific activity of the released purines was about four times higher than that of the purines retained in the slice but similar to that of the hypoxanthine in the slice. It is suggested that the purine release induced by electrical field stimulation differs in important respects from the release of a traditional neurotransmitter such as NA and that it depends on the intracellular formation of adenosine from a labile fraction of adenine nucleotides that is affected by neurotransmission.
Cerebral Ammonia Metabolism in Hyperammonemic Rats
Authors: Cooper åArthur J. L. , Mora Sol N. , Cruz Nancy F. , Gelbard Alan S.
Published: October 2006

The short‐term metabolic fate of blood‐borne [13N]ammonia was determined in the brains of chronically (8‐ or 14‐week portacaval‐shunted rats) or acutely (urease‐treated) hyperammonemic rats. Using a “freezeblowing” technique it was shown that the overwhelming route for metabolism of blood‐borne [13N]ammonia in normal, chronically hyperammonemic and acutely hyperammonemic rat brain was incorporation into glutamine (amide). However, the rate of turnover of [13N]ammonia to L‐[amide‐13N]glutamine was slower in the hyperammonemic rat brain than in the normal rat brain. The activities of several enzymes involved in cerebral ammonia and glutamate metabolism were also measured in the brains of 14‐week portacaval‐shunted rats. The rat brain appears to have little capacity to adapt to chronic hyperammonemia because there were no differences in activity compared with those of weight‐matched controls for the following brain enzymes involved in glutamate/ammonia metabolism: glutamine synthetase, glutamate dehydrogenase, aspartate aminotransferase, glutamine transaminase, glutaminase, and glutamate decarboxylase. The present findings are discussed in the context of the known deleterious effects on the CNS of high ammonia levels in a variety of diseases.
Axonal Transport of Polyamines in Intact and Regenerating Axons of the Rat Sciatic Nerve
Authors: Lindquist T. D. , Sturman J. A. , Gould R. M. , Ingoglia N. A.
Published: October 2006

The axonal transport of putrescine or its polyamine derivatives spermidine or spermine is a subject of some debate. We investigated this question by injecting [3H]putrescine into the lumbar spinal cord of the rat and measuring the accumulation of radioactivity central to ligatures placed on intact and regenerating sciatic nerves. In normal nerves, approximately twice as much radio activity built up proximal to these ligatures 2 or 3 days after injection than at more distal ligatures used to control for accumulation of radioactivity which might be due to tissue damage alone. In regenerating nerves the amount of radioactivity accumulating at the ligature was approx imately five times that at the distal ligature and two to three times greater than in intact nerves. The identity of the radioactivity in regenerating nerves, determined on an amino acid analyzer, was found to be primarily spermidine and an unknown compound that migrated as a frontal elution peak. Autoradiographic analysis showed that the radioactivity was largely confined to axons, but a significant amount of the silver grains was associated with Schwann cells and myelin sheaths surrounding labeled axons in both intact and regenerating nerves. The data indicate that polyamine derivatives of putrescine are transported axonally in rat sciatic nerves, and some of this transported material accumulates in Schwann cells surrounding the labeled axons. These processes are apparently augmented during regeneration of the injured axons.
Remodeling and Sorting Process of Ethanolamine and Choline Glycerophospholipids During Their Axonal Transport in the Rabbit Optic Pathway
Authors: Alberghina M. , Viola M. , Moro F. , Giuffrida A. M.
Published: October 2006

Abstract: The existence of a mechanism by which the ester‐ and ether‐linked aliphatic chains of the major phospholipids are retailored during their axonal transport and sorted to specific membrane systems along the optic nerve and tract was investigated. A mixture of [I‐14C]hexadecanol and [3H]arachidonic acid was injected into the vitreous body of albino rabbits. At 24 h and 8 days later, the distribution (as measured by the 3H/14C ratio) and the positioning (as monitored by hydrolytic procedures) of radioactivity in the various phospholipid classes of retina, purified axons, and myelin of the optic nerve and tract were determined. At the two intervals after labeling, the 3H/14C ratios of each diradyl type of phosphatidylethanolamine and phosphatidylcholine were (a) substantially unchanged all along the axons within the optic nerve and tract and (b) markedly modified in comparison with those found in the retina and axons for molecular species selectively restricted to myelin sheath. Evidence is thus available that intraxonally moving ethanolamine and choline glycerophospholipids, among others, are added to axonal membranes most likely without extensive modifications. In contrast, they are transferred into myelin after retailoring. Through these two processes, the sorting and targeting of newly synthesized phospholipids to their correct membrane domains. such as axoplasmic organelles, axolemma, or periaxonal myelin, could be controlled.
Differential Effects of Lithium on Muscarinic Receptor Stimulation of Inositol Phosphates in Rat Cerebral Cortex Slices
Authors: Batty Ian , Nahorski Stefan R.
Published: October 2006

Abstract: The accumulation of labelled inositol mono‐, bis‐, and trisphosphate in rat cerebral cortex slices was examined following preincubation with [3H]inositol. The muscarinic receptor agonist carbachol produced a rapid and sustained increased accumulation of each labelled inositol phosphate both in the presence and absence of 5 mM lithium. Lithium potentiated carbachol‐stimulated accumulation of inositol monophosphate (EC50 0.5 mM) and inositol bisphosphate (EC50 4 mM) in a concentration‐dependent manner. However, exposure to lithium in the presence of the muscarinic agonist produced a concentration‐ and time‐dependent inhibition of inositol trisphosphate accumulation that was not related to receptor desensitisation. Although the present data do suggest that polyphosphoinositides are substrates for agonist‐stimulated phospholipase C in brain, these results may not be entirely consistent with the production of inositol mono‐ and bisphosphate through inositol trisphosphate dephosphorylation. Furthermore, these data suggest site(s) additional to inositol monophosphatase that are affected by lithium.
Synthesis of a Stress Protein Following Transient Ischemia in the Gerbil
Authors: Nowak Thaddeus S.
Published: October 2006

Abstract: In vitro translation products of gerbil brain preparations, obtained from animals killed during recirculation following transient ischemia, showed increased synthesis of a 70‐kilodalton stress protein, identified by two‐dimensional gel electrophoresis. Stimulation of stress protein synthesis was evident as early as 2 h after recirculation, at which time overall translation activity remained low. Expression of the 70‐kilodalton protein reached a maximum at 8 h recirculation, when incorporation into other translation products had returned to essentially control levels. Increased incorporation into the stress protein was still detectable after 24 h recirculation. Although the functional consequences of increased expression of this stress protein remain unknown, these results suggest that the gerbil ischemia model may provide a useful experimental system in which to study the involvement of this phenomenon in processes related to postischemic cell damage and recovery.