bluuebunny/crossref_metadata_2025 huggingface dataset:
Calcium‐Dependent Release of Tyrosine in Brain Elicited by Stimulation of Neuropeptide Receptors
Authors: Arnerić Stephen P. , Meeley Mary P. , Reis Donald J.
Published: October 2006

Abstract: We sought to establish whether the endogenous opiate‐receptor agonist Met‐enkephalin (m‐ENK) selectively modulates the release of endogenous tyrosine (Tyr) from brain slices prepared from the corpus striatum (CS). Amino acids (AAs) released from slices of CS and, for comparison, cerebral cortex (Cx) were measured by HPLC. Incubation of slices with m‐ENK (1‐10 μM) increased the basal release of Tyr (up to 293% of control) from CS, but not Cx, whereas other nonneurotransmitter AAs, phenylal‐anine (Phe) and valine (Val), were unchanged. The release of the putative neurotransmitter AAs glutamate (Glu), tau‐rine (Tau), and glycine (Gly) were similarly increased by 50–150% with m‐ENK in slices of CS, but not Cx. The enhanced release of AAs by m‐ENK was prevented by removal of extracellular Ca2+ or by preincubation with the opiate receptor antagonist naloxone. Neuronal depolarization by potassium (5–55 mM) in the presence of Ca2+ did not affect the release of Tyr, whereas release of neurotransmitter AAs such as γ‐aminobutyric acid (GABA) were markedly increased. The increase in basal Tyr release by m‐ENK was not the result of a decreased uptake of Tyr. Relative to slices, the basal release of Tyr, Phe, and Val from a synaptosomal (P2) preparation of CS was small (8–51%) compared to that of GABA, Gly, Glu, and Tau (49–123%). Nonetheless, m‐ENK (10 μM) markedly increased the release of Tyr (to 833%), but not Glu, Gly, and Tau from the P2 fraction. Other neuropeptides including cholecystokinin octapeptide (CCK‐8), thyrotropin‐releasing hormone (TRH), and vaso‐active intestinal peptide (VIP) facilitated the release of Tyr from brain slices in a regionally specific pattern. We conclude that: (1) Tyr is released from nerve terminal‐enriched preparations of CS, but not Cx, by m‐ENK via an opiate receptor‐mediated, Ca2+‐dependent process with regional selectivity; (2) neuronal depolarization alone, however, does not affect the release of Tyr; (3) CCK‐8, TRH, and VIP also increase Tyr release with regional specificity, suggesting that receptors for other neuropeptides may also modulate Tyr release. The specific neuronal source and functional role of Tyr released from elements of CS via activation of opiate receptors remain to be elucidated.
Molecular Cloning of cDNA to mRNA for a Cerebellar Spot 35 Protein
Authors: Yamakuni T. , Kuwano R. , Odani S. , Miki N. , Yamaguchi K. , Takahashi Y.
Published: October 2006

Abstract: The nucleotide sequence of mRNA for rat cerebellar spot 35 protein, a Ca‐binding protein, was determined from recombinant complementary DNA (cDNA) clones. The sequence was composed of 1, 714 base pairs (bp) which included the 783 bp of the complete coding region, the 130 bp of the 5′‐noncoding region, and the 801 bp of the 3‐noncoding region containing a polyadenylation signal. In addition, a polyadenylic acid [poly(A)] tail was also found. Because the size of spot 35 mRNA was estimated to be about 1,900 bases by Northern blot analysis, the longest insert was verified to contain a nearly full‐length cDNA sequence including the poly(A) tail. The amino acid sequence of the protein deduced from the nucleotide sequence contains 261 amino acids and at least five Ca‐binding domains. There was a high homology in the amino acid sequences (79%) and the nucleotide sequences (77%) between spot 35 protein and chick intestinal Ca‐binding protein (28K).
Phosphatidylethanol Formation via Transphosphatidylation by Rat Brain Synaptosomal Phospholipase D
Authors: Kobayashi Mutsuhiro , Kanfer Julian N.
Published: October 2006

Abstract: Phosphatidylethanol (Peth) formation catalyzed by the transphosphatidylation activity of phospholipase D was demonstrated to occur in a rat brain synaptosomal enriched preparation. The optimal pH was determined to be 6.5, and the optimal ethanol concentration was determined to be 0.3–0.4 M with an apparent Km of 0.2 M. Peth formation was barely detectable in the absence of an appropriate activator and several unsaturated fatty acids were found to be effective activators. The concentrations of oleic acid required for maximum activation varied with the concentration of exogenous phosphatidylcholine present in the incubation mixtures. All detergents tested were significantly less active than the unsaturated fatty acids and divalent ions were not required for Peth formation. Phosphatidylcholine was the most effective phosphatidyl donor of the phospho‐lipids tested. Peth forming activity was greatest in the synap‐tic membrane fraction of the various brain subfractions examined. The 12,000 g‐100,000 g paniculate fraction of lung, heart, and adipose tissue had activities similar to that of brain.
Neurochemical Characteristics of a Novel Dorsal Root Ganglion X Neuroblastoma Hybrid Cell Line, F‐11
Authors: Francel Paul C. , Harris Kathy , Smith Michelle , Fishman Mark C. , Dawson Glyn , Miller Richard J.
Published: October 2006

Abstract: We investigated the properties of the novel dorsal root ganglion (DRG) hybrid cell line F‐11 to see how closely these cells resembled normal DRG cells. Under normal growth conditions, F‐11 cells appeared to contain several short neurite‐like processes. However, these cells could also be grown under conditions in which they showed a much more extensive neuronal morphology, exhibiting many long neurites. Several differentiated features of DRG cells were present on F‐11 cells. These included the presence of δ5‐opioid receptors, receptors for prostaglandins and brady‐kinin, and dihydropyridine‐sensitive calcium channels. F‐11 cells also synthesized and released a substance P‐Iike compound, as determined by immunoreactivity. Both the number of bradykinin receptors and the voltage‐sensitive calcium influx increased on cell differentiation. Opioid agonists (δ‐specificity) were found to decrease cyclic AMP levels in F‐l 1 cells in a naloxone‐ and pertussis toxin‐reversible fashion. Bradykinin stimulated the synthesis of inositol‐1,4‐bisphosphate and inositol‐1,4,5‐trisphosphate. Ca2+ channel agonists stimulated voltage‐sensitive Ca2+ influx in a dose‐dependent, stereospecific manner, whereas Ca2+ channel antagonists inhibited Ca2+ influx. F‐ll cells should, therefore, prove useful as models for authentic DRG neurons.
The Significance of Homovanillic Acid and 3,4‐Dihydroxyphenylacetic Acid Concentrations in Human Lumbar Cerebrospinal Fluid
Authors: Ebinger Guy , Michotte Yvette , Herregodts Patrick
Published: October 2006

Abstract: The concentrations of the acidic dopamine (DA) catabolites homovanillic acid (HVA) and 3,4‐dihydroxy‐phenylacetic acid (DOPAC) measured in human CSF are supposed to reflect the “turnover” of DA in the brain. The notion of “turnover” is, however, not synonymous with impulse nerve activity in the dopaminergic systems. Significant amounts of DOPAC and HVA could, indeed, be demonstrated in brain structures wherein dopaminergic inner‐vation has not been documented. It must also be noted that DA is not only a neurotransmitter itself, but also a precursor of norepinephrine and epinephrine. Furthermore, in lumbar CSF, levels of biogenic amine catabolites partially reflect metabolism in the spinal cord and may have limited relevance to neurotransmission in the brain. To elucidate these points further, we determined the concentrations of DOPAC and HVA in 22 areas of six human brains and eight levels of six human spinal cords. The data were correlated with the concentration of DA. Quantitative determinations were done using HPLC with electrochemical detection, after solvent and ion‐pair extraction. In this study, significant amounts of both DOPAC and HVA were demonstrated in brain structures not previously associated with dopaminergic innervation. The relatively lower DA concentration in these structures suggests that in these regions, the DOPAC and HVA concentrations are unrelated to dopaminergic neurotransmission. The possible role of capillary walls and glial cells in the catabolism of DA must be further evaluated. The demonstration of DOPAC and HVA in the spinal cord is another argument against the hypothesis that CSF levels of HVA and DOPAC reflect closely the activity of the dopaminergic systems in the brain.
Calcium Dependence of Muscarinic Receptor‐Mediated Catecholamine Secretion from the Perfused Rat Adrenal Medulla
Authors: Harish Orna E. , Kao Lung‐Sen , Raffaniello Robert , Wakade Arun R. , Schneider Allan S.
Published: October 2006

Abstract: It had previously been thought that muscarinic cholinergic receptors utilize an influx of extracellular calcium for activation of adrenomedullary catecholamine secretion. However, it has recently been demonstrated that muscarinic receptors on isolated adrenal chromaffin cells can elevate cytosolic free calcium levels in a manner independent of extracellular calcium, presumably by mobilizing intracellular calcium stores. We now demonstrate that muscarinic receptor‐mediated catecholamine secretion from perfused rat adrenal glands can occur under conditions of extracellular calcium deprivation that are sufficient to block both nicotine‐ and electrically stimulated release. Three independent conditions of extracellular calcium deprivation were used: (a) nominally calcium‐free perfusion solution (no calcium added), (b) EGTA‐containing calcium‐free perfusion solution, and (c) perfusion solution containing the calcium channel blocker verapamil. Secretion was evoked from the perfused glands by either transmural electrical stimulation or injection of nicotine or muscarine into the perfusion stream. Each condition of calcium deprivation was able to block nicotine‐ and electrically stimulated catecholamine release in an interval that left muscarine‐evoked release largely unaffected. The above results demonstrate that muscarine‐evoked catecholamine secretion from perfused rat adrenal glands can occur in the absence of extracellular calcium, presumably by mobilization of intracellular calcium. The latter may be due to muscarinic receptor‐mediated generation of inositol trisphosphate.
Characterization of the Actions of Toxins II‐9.2.2 and II‐10 from the Venom of the Scorpion Centruroides noxius on Transmitter Release from Mouse Brain Synaptosomes
Authors: Sitges María , Possani Lourival Domingos , Bayón Alejandro
Published: October 2006

Abstract: Toxic peptides II‐9.2.2 and II‐10, purified from Centruroides noxius venom, bear highly homologous N‐ter‐minal amino acid sequences, and both toxins are lethal to mice. However, only toxin II‐10 is active on the voltage‐clamped squid axon, selectively decreasing the voltage‐dependent Na+ current. Here, we have tested toxins II‐9 and II‐10 on synaptosomes from mouse brain: both toxins increased the release of γ‐[3H]aminobutyric acid ([3H]GABA). Their effect was completely blocked by tetro‐dotoxin or by the absence of external Na+. Also, both toxins increased Na+ permeability in isolated nerve terminals. Besides the observation that toxin II‐9 is active on synaptosomes, the effect of toxin II‐10 in this preparation is opposite to that observed in the squid axon. Thus, our results reflect functional differences between the populations of Na+ channels in mouse brain synaptosomes and in the squid axon. The release of GABA evoked by these toxins from synaptosomes required external Ca2+ and was blocked by Ca2+ channel blockers (verapamil and Co2+). This latter observation is in sharp contrast to the releasing action of veratrine, which evoked release even in the absence of external Ca2+. Furthermore, the action of both C. noxius toxins was potentiated by veratrine, a result suggesting they have different mechanisms of action. Among drugs that release neuro‐transmitters by increasing Na+ permeability, it is noteworthy that scorpion toxins are the only ones yet reported to have a strict requirement for external Ca2+.
Acetylcholinesterase from the Skeletal Muscle of the Lamprey Petromyzon marinus Exists in Globular and Asymmetric Forms
Authors: Pezzementi Leo , Reinheimer Ellen J. , Pezzementi Maureen L.
Published: October 2006

Abstract: To obtain information about the evolution of acetylcholinesterase (AChE), we undertook a study of the enzyme from the skeletal muscle of the lamprey Petromyzon marinus, a primitive vertebrate. We found that the cholines‐terase activity of lamprey muscle is due to AChE, not pseudocholinesterase; the enzyme was inhibited by 1,5‐bis(4‐allyldimethylammonium phenyl) pentane‐3‐one (BW284C51), but not by tetramonoisopropyl pyrophos‐phortetramide (iso‐OMPA) or ethopropazine. Also, the enzyme had a high affinity for acetylthiocholine and was inhibited by high concentrations of substrate. A large fraction of the AChE was found to be glycoprotein, since it was precipitated by concanavalin A‐agarose. Optimal extraction of AChE was obtained in a high‐salt detergent‐containing buffer; fractional amounts of enzyme were extracted in buffers lacking salt and/or detergent. These data suggest that globular and asymmetric forms of AChE are present. On sucrose gradients, enzyme that was extracted in high‐salt detergent‐containing buffer sedimented as a broad peak of activity corresponding to G4; additionally, there was usually a peak corresponding to A12. Sequential extraction of AChE in conjunction with velocity sedimentation resolved minor forms of AChE and revealed that the G1, G2, G4, A4, A8, and A12 forms of AChE cpuld be obtained from the muscle. The identity of the forms was confirmed through high‐salt precipitation and collagenase digestion. The asymmetric forms of AChE were precipitated in low ionic strength buffer, and their sedimentation coefficients were shifted to higher values by collagenase digestion. We discuss these results in terms of the evolution of AChE in deuterostome vertebrates and invertebrates and in terms of the physiological significance of asymmetric forms of AChE for muscle function.
Evidence for an Involvement of Membrane Lipids in the Control of Neuronal Nicotinic Receptor Function Using Bungarotoxin II‐S1
Authors: Quik M.
Published: October 2006

Abstract: Previous work has shown that a toxin fraction, bungarotoxin (BGT) II‐S1, isolated from Bungarus multi‐cinctus venom could inhibit nicotinic receptor‐mediated function. Experimental evidence suggested that this effect of the toxin might be due to a direct interaction of the toxin at the acetylcholine binding site and/or to its phospholipase activity. The toxin's enzymic activity has been further characterized; it has phospholipase activity of the A2 type with a Vmax of 12 pmol/min/ng protein and a Km of 300 μM. Phospholipases can produce their effects on a tissue through a variety of mechanisms including the disruption of important lipid protein bonds or the production of free fatty acids which interact with the tissue. To test for this latter possibility, various concentrations of fatty acid‐free bovine serum albumin were added to the incubation medium. Fatty acid‐free bovine serum albumin partially reversed the inhibition of carbachol‐stimulated 1‐[1,2‐3H(N)]amino‐4‐guanidobu‐tane ([3H]agmatine) uptake (used as a measure of ion flux) into the ganglion produced by BGT II‐S1 (1.0 μM). In an attempt to determine which fatty acids might be responsible for this effect, various fatty acids were added to the incubation medium and their effect on nicotinic receptor‐mediated [3H]agmatine uptake determined. Arachidonic acid decreased amine uptake by approximately 50% over the control carbachol‐stimulated uptake; linoleic and oleic acid, on the other hand, did not significantly affect the response. This observation could imply that arachidonic acid is the fatty acid produced by the action of BGT II‐S 1 on the tissue to mediate the toxin's inhibitory effect. These results could suggest that alterations in the lipid environment of the neuronal nicotinic receptor may serve as a regulatory mechanism to control function.
Alterations in Lipid Metabolism, Na+,K+‐ATPase Activity, and Tissue Water Content of Spinal Cord Following Experimental Traumatic Injury
Authors: Faden Alan I. , Chan Pak H. , Longar Susan
Published: October 2006

Abstract: Traumatic spinal cord injury has recently been shown to cause a rapid increase in free fatty acids (FFAs) and lipid degradation in cats. The present studies report a more delayed, time‐dependent increase in FFAs and a concomitant decrease in phospholipids following traumatic spinal injury in rats. The largest percentage increases were found for polyunsaturated fatty acids, particularly arachi‐donic acid. Associated with these changes were a reduction in the activity of Na+,K+‐ATPase and development of spinal cord edema. These findings support the hypothesis that traumatic spinal cord injury leads to delayed, as well as early, hydrolysis of membrane phospholipids, resulting in the liberation of FFAs. Such changes may contribute to secondary spinal cord injury either through direct effects on membranes or through the actions of secondary metabolic products such as the eicosanoids. The latter may cause tissue injury by contributing to the reduction in spinal cord blood flow or through inflammatory responses that follow trauma.
Mass Fragmentographic Determination of γ‐Aminobutyric Acid and Glutamic Acid in Discrete Amygdaloid Nuclei of Rat Brain
Authors: Nakamura Kaoru , Matsumoto Takashi , Hirano Makoto , Kagoshima Hiroshi , Kuroki Toshihide , Yao Hiroshi , Uchimura Hideyuki , Nakahara Tatsuo
Published: October 2006

Abstract: A mass fragmentographic method for the simultaneous quantification of γ‐aminobutyric acid (GABA) and glutamic acid is described. In a convenient one‐step reaction, the two ammo acids were derivatized with pentafluor‐opropionic anhydride and pentafluoropropanol. The deri‐vatization products were stable for several days. The technique has been applied to the assay of GABA and Glu in five amygdaloid nuclei of the rat brain. The GABA level was high in the central and medial nuclei, whereas the Glu level was high in the lateral and basal nuclei. The regional distribution of GABA was different from that of Glu within the amygdaloid nuclei.
Evidence for Somatostatin‐Containing Fibers Projecting from the Pallidal Complex to the Striatum of the Rat
Authors: Widmann R. , Mensdorff‐Pouilly N. , Pfaller K. , Sperk G.
Published: October 2006

Abstract: The origin of afferent somatostatin‐containing fibers terminating in medial and ventral parts of the striatum has been investigated by performing various neuro‐chemical and surgical lesions in the rat. Lesions of the anterior hypothalamus, amygdala, and the hippocampal commissure as well as lesions with 6‐hydroxydopamine and 5,7‐dihydroxytryptamine failed to decrease striatal soma‐tostatin levels. However, thermal coagulation of the globus pallidus or knife‐cut lesions performed ventrally to the striatum resulted in significant decreases in striatal somatostatin content. Analysis of the topographical distribution of somatostatin within the striatum after thermal lesions of the globus pallidus as well as after kainic acid‐induced seizures revealed a preferential loss of the peptide in medial and ventral portions of the striatum, the site of terminating afferent somatostatin nerve fibers. The data suggest that the striatal afferent somatostatin‐containing neurons may originate in the area of the globus pallidus.
Subcellular Distribution of Ependymins in Goldfish Brain Measured by Radioimmunoassay
Authors: Schmidt Rupert , Lapp Harald
Published: October 2006

Abstract: Goldfish CNS was fractionated by differential and density gradient centrifugation. The fractions obtained were characterized by marker enzymes typical of various subcellular organelles. They were further analyzed by radioimmunoassay for their contents of ependymins, two CNS glyco‐proteins known to participate in biochemical reactions after learning events. Ependymins were shown to be major constituents of the soluble cytoplasm (5.6% of the total protein content). The nuclear fraction was virtually devoid of ependymins (0.6% of protein). Small amounts were observed in the crude synaptosomal and microsomal fractions (1.0 and 3.5%, respectively). The highest steady‐state concentration of ependymins, however, was measured in the brain extracellular fluid (15.6% of the protein), including the CSF. The specificity of the distribution was examined by intracerebro‐ventricular injection of 125I‐labeled ependymins as exogenous marker substances. No indication of an artificial redistribution of the radiolabel during homogenization and frac‐tionation was obtained. The exogenous analogues of ependymins were, however, incorporated in vivo into organelles recovered in the nuclear and crude synaptosomal fractions. Our results suggest that ependymins may interact with synaptic membranes from the extracellular fluid, although so far no evidence for a specific receptor‐type binding site could be obtained in vitro.
Changes in Subcellular Distribution of Ependymins in Goldfish Brain Induced by Learning
Authors: Schmidt Rupert
Published: October 2006

Abstract: Goldfish were trained for 4 h to swim with an attached polystyrene foam float and tested for retention 3 days later. Intracerebroventricular injection of anti‐ependymin antisera was shown to prevent long‐term memory formation of this vestibulomotor learning task, as reported previously. In further experiments, fish were killed 4–14 h after the start of training. The brains were dissected, incubated in an isoosmolar solution for collection of proteins of the brain extracellular fluid (ECF), homogenized, and fractionated by differential centrifugation. The ECF, a supernatant fraction enriched in cytoplasmic constituents (S3), and various par‐ticulate subcellular fractions were analyzed for their epen‐dymin contents by radioimmunoassay. No statistically significant changes that might be induced by the learning were revealed in any of the participate fractions. Steady‐state concentrations of ependymins in the cytoplasm, however, increased temporarily by 39% in fish that had mastered the training task as compared with nonlearning animals (passive and active controls). In the ECF, the specific concentration of ependymins first decreased to 88% of control levels (4–5 h after the start of training), but later on, it increased to 138% (8–14 h). Apparently, ependymins present in the ECF are used during biochemical reactions of memory consolidation. The resulting decrease in extracellular epen‐dymin concentrations might trigger their resynthesis in the cytoplasm and lead to an increased release of these glyco‐proteins into the ECF.
Effect of N‐Methyl‐4‐Phenylpyridinium Ion on Monoamine Oxidase in a Clonal Rat Pheochromocytoma Cell Line, PC 12h
Authors: Naoi Makoto , Suzuki Hiroko , Kiuchi Kazutoshi , Takahashi Tsutomu , Nagatsu Toshiharu
Published: October 2006

Abstract: The effects of the neurotoxin N‐methyl‐4‐phenylpyridinium ion (MPP+) on the enzymes involved in synthesis and catabolism of catecholamines were examined using a clonal rat pheochromocytoma cell line, PC12h, as a model of dopaminergic neurons. MPP+ added in the culture medium was found to be accumulated in PC12h cells after 30‐min incubation. Monoamine oxidase (MAO) activity in PC12h cells was inhibited by MPP+ in a dose‐dependent way from 10 nM to 10 μM, but concentrations of MPP+ higher than 100 μM were found to increase the MAO activity. At the lower concentrations MPP+ inhibited MAO non‐competitively with respect to the substrate, kynuramine, and at the higher concentrations it increased both the Km and the Vmax values of MAO toward the substrate. On the other hand, tyrosine hydroxylase activity and the dopamine concentrations in PC 12 cells were not changed by incubation with MPP+ for 30 min, 60 min, or 24 h.
Excitatory Amino Acid Receptors and Depolarization‐Induced Ca2+ Influx into Hippocampal Slices
Authors: Crowder J. M. , Croucher M. J. , Bradford H. F. , Collins J. F.
Published: October 2006

Abstract: Hippocampal brain slices were incubated with depolarizing agents or excitatory amino acids either alone or in the presence of excitatory amino acid antagonists [ω‐phosphonic α‐aminocarboxylic acids—2‐amino‐4‐phosphonobutyric acid (AP4), 2‐amino‐5‐phosphonovaleric acid (AP5), or 2‐amino‐7‐phosphonoheptanoic acid (AP7)—or γ‐D‐glutamylaminomethylsulphonic acid (GAMS)] or a calcium‐channel blocker, (S)‐1‐(3‐methoxyphenyl)‐3‐methylaza‐7‐cyano‐7‐ (3,4‐dimethoxyphenyl)‐8‐methyl‐nonane hydrochloride [(‐)‐D888]. The uptake of 45Ca2+ and the efflux of glutamate or aspartate induced by veratrine or high K+ was blocked (54–76%) by AP7 (IC50 46–250 μM). AP5 and AP4 were less effective. (‐)‐D888 (10 μM) caused 100% block of evoked 45Ca2+ uptake. Uptake of 45Ca2+ induced by exogenous glutamate, aspartate, and N‐methyl‐D‐aspartate (NMDA) was also inhibited by AP7, whereas GAMS completely blocked the action of kainate and partially blocked that of glutamate. The action of NMDA in stimulating 45Ca2+ uptake was Mg2+‐sensitive, low Mg2+ levels in the incubation medium selectively enhancing the response. It is concluded that Ca2+ uptake evoked by excitatory amino acids is receptor‐mediated, and that released excitatory amino acids are responsible for a large part of the action of veratrine and high K+ in stimulating 45Ca2+ uptake.
Inhibition of Lactate Production in Rat Brain Extracts and Synaptosomes by 3‐[4‐(Reduced 3‐Pyridine Aldehyde‐Adenine Dinucleotide)]‐Pyruvate
Authors: Cooper Arthur J. L. , Lai James C. K. , Coleman Anton E. , Pulsinelli William A.
Published: October 2006

Abstract: In basic solutions, pyruvate enolizes and reacts (through its 3‐carbon) with the 4‐carbon of the nicotinamide ring of NAD+, yielding an NAD‐pyruvate adduct in which the nicotinamide ring is in the reduced form. This adduct is a strong inhibitor of lactate dehydrogenase, presumably because it binds simultaneously to the NADH and pyruvate sites. The potency of the inhibition, however, is muted by the adduct's tendency to cyclize to a lactam. We prepared solutions of the pyruvate adduct of NAD+ and of NAD+ analogues in which the —C(O)NH2 of NAD+ was replaced with C(S)NH2, −C(O)CH3, and −C(O)H. Of the four, only the last analogue, 3‐[4‐(reduced 3‐pyridine aldehyde‐adenine dinucleotide)]‐pyruvate (RAP) cannot cyclize and it was found to be the most potent inhibitor of beef heart and rat brain lactate dehydrogenases. The inhibitor binds very tightly to the NADH site (Ki∼ 1 nM for the A form). Even at high concentrations (20 μM), RAP had little or no effect on rat brain glyceraldehyde‐3‐phosphate, pyruvate, α‐ketoglutarate, isocitrate, soluble and mitochondrial malate, and glutamate dehydrogenases. The glycolytic enzymes, hexokinase and phosphofructokinase, were similarly unaffected. RAP strongly inhibited lactate production from glucose in rat brain extracts but was less effective in inhibiting lactate production from glucose in synaptosomes.
Characterization of the Quisqualate Receptor Linked to Phosphoinositide Hydrolysis in Neurocortical Cultures
Authors: Patel Jitendra , Moore W. Craig , Thompson Carolann , Keith Richard A. , Salama Andre I.
Published: October 2006

Abstract: Activation of phosphoinositide metabolism is an early event in signal transduction for a number of neurotransmitters and hormones. In primary cultures of rat neurocortical cells, various excitatory amino acids stimulate inositol phosphate production with a rank order of potency of quisqualate > ibotenate > glutamate > kainate, N‐methyl‐d‐aspartate > α‐amino‐3‐hydroxyl‐5‐methyl‐4‐isoxazole propionate. This response to excitatory amino acids was insensitive to a variety of excitatory amino acid antagonists including 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione, 3–3(2‐carboxypiperazine‐4‐yl)propyl‐l‐phosphonate, and 2‐amino‐4‐phosphonobutyrate. The individual responses of quisqualate‐, ibotenate‐, and kainate‐stimulated inositol phosphate production were not additive. These results suggest that phosphoinositide metabolism activated by excitatory amino acids is mediated by a unique quisqualate‐preferring receptor that is not antagonized by known N‐methyl‐d‐aspartate and non‐N‐methyl‐d‐aspartate antagonists, and is relatively insensitive to α‐amino‐3‐hydroxyl‐5‐methyl‐4‐isoxazole propionate.
Extracellular Striatal Dopamine and Its Metabolites During Transient Cerebral Ischaemia
Authors: Obrenovitch Tihomir Paul , Sarna Gurcharan S. , Matsumoto Takashi , Symon Lindsay
Published: October 2006

Abstract: Severe cerebral ischaemia has been repeatedly shown to provoke a massive increase in striatal extracellular dopamine (DA). These experiments were undertaken to determine the duration of the DA increase produced by transient ischaemia, and the fate of the released DA during recirculation. Experiments were performed in anaesthetised rats subjected to 20 min of cerebral ischaemia, followed by 80 min of reperfusion, before cardiac arrest. Measurements of catechols were made in the striatum using in vivo differential pulse voltammetry (DPV), each 4 min, throughout the experiment and for 60 min after cardiac arrest. DPV data were substantiated with intracerebral dialysis; 20‐min dialysate samples were analysed for DA and homovanillic acid (HVA) using HPLC. In 6 of 11 rats, ischaemia induced a massive DA release in the striatum, resulting in a marked increase in extracellular levels (350–1,200%), which persisted throughout ischaemia. DPV and intracerebral dialysis demonstrated that DA was totally cleared from the extracellular space within minutes of reperfusion, whereas both its acidic metabolites (3,4‐dihydroxyphenylacetic acid and HVA) increased slightly. These results indicate that DA released during 20‐min ischaemia is rapidly cleared during reperfusion, mainly via reuptake. In the five other rats, only a relatively small and transient increase in the DPV catechol peak was detectable, cleared before the end of ischaemia, probably reflecting less severe ischaemia; small or no changes were detectable in the corresponding dialysate. The latter data suggest that different change(s) in the nigrostriatal dopaminergic system may occur, according to the severity of ischaemia.
Effect of Climbing Fiber Deprivation on Release of Endogenous Aspartate, Glutamate, and Homocysteate in Slices of Rat Cerebellar Hemispheres and Vermis
Authors: Vollenweider F. X. , Cuénod M. , Do K. Q.
Published: October 2006

Abstract: Aspartate (Asp) and/or glutamate (Glu) have been proposed as putative excitatory transmitters released from synaptic terminals of the olivo‐cerebellar climbing fiber afferents to the Purkinje cells. Investigations of the climbing fiber transmitter(s) separately for hemispheres and vermis were performed to examine whether the current controversy over the role of Asp as a neurotransmitter in the climbing fibers may be due to topographic differences. K+‐induced Ca2+‐dependent release of endogenous substances was investigated in slices of cerebellar hemisphere and vermis of control rats and those deprived of climbing fibers by 3‐acetylpyridine (3‐AP) treatment. A release of Asp and Glu, as well as a small but significant release of homocysteic acid (HCA) was confirmed in control rats. Climbing fiber deprivation by 3‐AP treatment reduced the stimulated release of Asp by 48% in slices of cerebellar hemispheres, but not in vermis. Climbing fiber deprivation completely abolished the release of HCA in both hemispheres and vermis. The release of HCA, Asp, and Glu from slices of control and climbing fiber‐deprived rats evoked by 50 mMK+ was >90% Ca2+‐dependent. These results support the hypothesis that Asp is a transmitter candidate of the climbing fibers projecting to the cerebellar hemispheres, but not to the vermis, and provide the first evidence that HCA can be linked to a specific pathway.
The Glycine Site of the N‐Methyl‐D‐Aspartate Receptor Channel: Differences Between the Binding of HA‐966 and of 7‐Chlorokynurenic Acid
Authors: Kloog Yoel , Lamdani‐Itkin Hadas , Sokolovsky Mordechai
Published: October 2006

Abstract: The mechanisms of action of three different glycinesite antagonists of the N‐methyl‐D‐aspartate (NMDA)‐receptor channel were analyzed employing [3H]glycine direct binding assays, as well as functional glycine‐ and glutamate‐induced uncompetitive blocker binding assays. The latter assays measure apparent channel opening. All three antagonists tested, viz., 7‐chlorokynurenic acid (7‐Cl‐KYNA), kynurenic acid (KYNA), and 1‐hydroxy‐3‐aminopyrrolidone‐2 (HA‐966), inhibited the binding of [3H]glycine to the NMDA receptor in a dose‐dependent manner. These antagonists also inhibited the glycine‐induced increase in accessibility of the uncompetitive blocker [3H]N‐[1‐(2‐thienyl)cyclohexyl]‐piperidine ([3H]TCP) to the channel. 7‐Cl‐KYNA and KYNA, but not HA‐966, completely blocked the glutamate‐induced binding of [3H]TCP, in a manner similar to the non‐competitive manner in which the selective NMDA antagonist D‐(‐)‐2‐amino‐5‐phosphonovaleric acid (AP‐5) inhibited glycine‐induced [3H]TCP binding. The inhibitory effects of HA‐966 and of AP‐5 on glutamate‐induced [3H]TCP binding were overcome when glutamate concentrations were increased. Of the three antagonists, 7‐Cl‐KYNA appears to be the most potent (Ki= 0.4–1.0 μM) and the most selective glycine antagonist. KYNA was found to act at both the glycine (Ki= 40–50 μM) and the glutamate sites. In contrast, HA‐966 (Ki= 6–17 μM) appears to act either on a domain distinct from the glutamate and the glycine sites, but tightly associated with the latter, or at the glycine site, but according to a mechanism distinct from that of 7‐Cl‐KYNA. The results also show that simultaneous occupancy of the glycine and the glutamate sites of the NMDA receptor is essential for their functional activation, and support two concepts suggested by previous electrophysiological experiments: that glycine is a co‐agonist of glutamate, and that glycine is at least an M‐type allosteric effector of glutamate (i.e., it increases the maximal glutamate response without affecting its apparent binding constant).
Guanine Nucleotide‐Binding Protein Regulation of Microsomal Phospholipase D Activity of Canine Cerebral Cortex
Authors: Qian Zhuo , Reddy Padala V. , Drewes Lester R.
Published: October 2006

Abstract: The hydrolytic activity of microsomal phospholipase D from canine cerebral cortex was measured by a radiochemical assay using 1,2‐dipalmitoyl‐sn‐glycerol‐3‐phosphoryl[3H]choline and 1‐palmitoyl‐2‐[9, 10(n)‐3H]palmitoyl‐sn‐glycerol‐3‐phosphorylcholine as the exogenous substrates. Of several detergents tested, Triton X‐100 was found to be the most effective in allowing expression of phospholipase D hydrolytic activity. The microsomal phospholipase D does not require any metal ion for its hydrolytic activity. Calcium and magnesium were slightly inhibitory between concentrations of 1 and 4 mM, but zinc was greatly inhibitory, causing a loss of >90% activity at the 4 mM concentration. Nonhydrolyzable guanine nucleotide analogues such as guanosine 5′‐(3‐O‐thio)triphosphate and guanyl‐5′‐yl‐(β,γ‐methylene)diphosphonate but not guanosine 5′‐(2‐thio)diphosphate were able persistently to stimulate phospholipase D hydrolytic activity at micromolar concentrations. Guanosine 5′‐(2‐thio)diphosphate was capable of partially blocking guanosine 5′‐(3‐O‐thio)triphosphate stimulation of phospholipase D. Aluminum fluoride was also able to cause a two‐ to threefold increase in hydrolytic activity of the phospholipase D. Cholera toxin had a stimulatory effect on the hydrolytic activity of phospholipase D, whereas islet‐activating protein pertussis toxin had no effect. These results indicate that regulation of microsomal phosphatidylcholine phospholipase D activity by the guanine nucleotide‐binding protein(s) in canine cerebral cortex may play an important role in signal transduction processes as well as in brain choline metabolism.
Production of 1,2‐Diacylglycerol in PC12 Cells by Nerve Growth Factor and Basic Fibroblast Growth Factor
Authors: Altin Joseph G. , Bradshaw Ralph A.
Published: October 2006

Abstract: The addition of nerve growth factor (NGF) or basic fibroblast growth factor (bFGF) to PC12 cells prelabeled with [3H]inositol and preincubated for 15 min in the presence of 10 mM LiCl stimulated the production of inositol phosphates with maximal increases of 120–180% in inositol monophosphate (IP), 130–200% in inositol bisphosphate (IP2), and 45–50% in inositol trisphosphate (IP3) within 30 min. The majority of the overall increase (approximately 85%) was in IP; the remainder was recovered as IP2 and IP3 (approximately 10% as IP2 and 5% as IP3). Under similar conditions, carbachol (0.5 mM) stimulated about a 10‐fold increase in IP, a sixfold increase in IP2, and a fourfold increase in IP3. The mass level of 1,2‐diacylglycerol (DG) in PC12 cells was found to be dependent on the incubation conditions; in growth medium [Dulbecco's modified Eagle's medium (DME) plus serum], it was around 6.2 mol %, in DME without serum, 2.5 mol %, and after a 15‐min incubation in Dulbecco's phosphate‐buffered saline, 0.62 mol %. The addition of NGF and bFGF induced an increase in the mass level of DG of about twofold within 1–2 min, often rising to two‐ to threefold by 15 min, and then decreasing slightly by 30 min. This increase was dependent on the presence of extracellular Ca2+, and was inhibited by both phenylarsine oxide (25 μM) and 5′‐deoxy‐5′‐methylthioadenosine (3 mM). Under similar conditions, 0.5 mM carbachol stimulated the production of DG to the same extent as 200 ng/ml NGF and 50 ng/ml bFGF. Because carbachol is much more effective in stimulating the production of inositol phosphates, the results suggest that both NGF and bFGF stimulate the production of DG primarily from phospholipids other than the phosphoinositides.
Selective Alterations in Presynaptic Cytomatrix Protein Organization Induced by Calcium and Other Divalent Cations That Modulate Exocytosis
Authors: Garner Judy A.
Published: October 2006

Abstract: Rises in intracellular calcium cause several events of physiological significance, including the regulated release of neuronal transmitters. In this study, the effects of divalent cations on the structural organization of cytomatrix in presynaptic terminals was examined. [35S]Methionine‐radiolabeled guinea pig retinal ganglion cell cytomatrix proteins were axonally transported [in slow component b (SCb) of axonal transport] to the neuron terminals in the superior colliculus. When the peak of radiolabeled cytomatrix proteins reached the terminals, synaptosomes containing the radiolabeled cytomatrix proteins were prepared. Approximately 40% of each SCb protein was soluble after hypoosmotic lysis of the radiolabeled synaptosomes in the presence of divalent cation chelators. Lysis of synaptosomes in the presence of calcium ions over a range of concentrations, however, caused a dramatic decrease in solubility of the presynaptic SCb proteins. The cytoplasmic effects may result from a calcium‐dependent condensation of cytoplasm around presynaptic terminal membrane systems. There are two major presynaptic SCb proteins (at 60 and 35 kDa), that exhibited exceptional behavior: they remained as soluble in the presence of calcium as under control conditions, suggesting that they were relatively unaffected by the mechanism causing the decrease in SCb protein solubility. Also examined were the effects of other alkaline earth and transition metal divalent cations on the presynaptic SCb proteins.
A Method for the In Vivo Investigation of the Serotonergic 5‐HT2 Receptors in the Human Cerebral Cortex Using Positron Emission Tomography and 18F‐Labeled Setoperone
Authors: Blin Jerôme , Sette Giuliano , Fiorelli Marco , Bletry Olivier , Elghozi Jean Luc , Crouzel Christian , Baron Jean Claude
Published: October 2006

Abstract: Following previous validation in baboons, we have studied the characteristics of [18F]setoperone as a radioligand for investigating serotonergic 5‐hydroxytryptamine2 (5‐HT2) receptors in the normal, unmedicated human brain with positron emission tomography (PET); subjects orally pretreated with therapeutic amounts of ketanserin, sulpiride, or prazosin were also studied to evaluate the specificity and sensitivity of [18F]setoperone brain specific binding. In controls (n = 10), the tracer showed a clear‐cut retention in both frontal cortex and striatum (known to contain a high density of 5‐HT2 receptors) relative to cerebellum (known to be devoid of 5‐HT2receptors). In the seven young controls (20–39 years old), the frontal cortex/cerebellum and striatum/cerebellum ratios increased during the first hour to reach similar values of 2.53 ± 0.12 and 2.38 ± 0.11 (mean ± SEM), respectively, and were essentially stable during the second hour. Pretreatment with ketanserin (a 5‐HT2 blocker) significantly reduced the frontal cortex/cerebellum ratio to 0.7–1.0 at 65 min, whereas the striatum/cerebellum ratio was significantly, but only partially, reduced. During sulpiride treatment (a D2 blocker), the frontal cortex/cerebellum ratio was not altered, whereas the striatum/cerebellum ratio was significantly, but only partially, reduced. With prazosin pretreatment (an α1‐adrenergic blocker), neither the frontal cortex/cerebellum nor the striatum/cerebellum ratio was modified. These data in humans with PET demonstrate that [18F]setoperone labels with high sensitivity and selectivity 5‐HT2 receptors in the frontal cortex; in striata, however, binding is to both 5‐HT2 and D2 receptors. The deproteinated‐to‐whole plasma radioactivity concentration ratio increased with time following injection. The mean percentage of intact [18F]setoperone, in deproteinated plasma, was 82, 74, 53, 45, 30, and 22% at 5, 10, 20, 30, 60, and 110 min following injection, respectively. These data indicate that [18F]setoperone (a) is significantly bound to plasma proteins and (b) is significantly metabolized into several labeled metabolites that are much more hydrophilic than setoperone and, hence, presumably do not cross the blood–brain barrier. These results suggest the suitability of [18F]setoperone data for modeling of 5‐HT2 receptor binding in brain.
Acute Effects of Typical and Atypical Antipsychotic Drugs on the Release of Dopamine from Prefrontal Cortex, Nucleus Accumbens, and Striatum of the Rat: An In Vivo Microdialysis Study
Authors: Moghaddam Bita , Bunney Benjamin S.
Published: October 2006

Abstract:In vivo microdialysis has been used to study the acute effects of antipsychotic drugs on the extracellular level of dopamine from the nucleus accumbens, striatum, and prefrontal cortex of the rat. (–)‐Sulpiride (20, 50, and 100 mg/kg i.v.) and haloperidol (0.1 and 0.5 mg/kg i.v.) enhanced the outflow of dopamine in the striatum and nucleus accumbens. In the medial prefrontal cortex, (–)‐sulpiride at all doses tested did not significantly affect the extracellular level of dopamine. The effect of haloperidol was also attenuated in the medial prefrontal cortex; 0.1 mg/kg did not increase the outflow of dopamine and the effect of 0.5 mg/kg haloperidol was of shorter duration in the prefrontal cortex than that observed in striatum and nucleus accumbens. The atypical antipsychotic drug clozapine (5 and 10 mg/kg) increased the extracellular concentration of dopamine in all three regions. In contrast to the effects of sulpiride and haloperidol, that of clozapine in the medial prefrontal cortex was profound. These data suggest that different classes of antipsychotic drugs may have distinct effects on the release of dopamine from the nigrostriatal, mesolimbic, and mesocortical terminals.
Interaction of Asymmetric and Globular Acetylcholinesterase Species with Glycosaminoglycans
Authors: Ramirez Galo , Barat Ana , Fernández Hugo L.
Published: October 2006

Abstract: Chicken muscle and retina, and rat muscle asymmetric acetylcholinesterase (AChE) species were bound to immobilized heparin at 0.4 M NaCl. Binding efficiency was between 50 and 80% for crude fraction I A‐forms (A1; muscle), and nearly 100% for fraction II A‐forms (A11; muscle and retina). Antibody‐affinity‐purified A1‐forms (chicken) were, however, quantitatively bound to heparin–agarose gels, whereas diisopropylfluorophosphate‐inactivated high‐salt extracts partially prevented the binding of both A1 and A11 AChE forms, thus suggesting the presence in crude A1 extracts of heparin‐like molecules interfering with the tail–heparin interaction. All bound A‐forms were progressively displaced from the heparin–agarose columns by increasing salt concentrations, with maximal release at about 0.6 M. They were also efficiently eluted by heparin solutions (1 mg/ml), other glycosaminoglycans being much less effective. Chicken globular AChE forms (G‐forms, both low‐salt‐soluble and detergent‐soluble) also bound to immobilized heparin in the absence of salt. Stepwise elution with increasing NaCl concentrations showed maximal release of G‐forms at 0.15 M, all globular forms being totally displaced from the column at 0.4 M NaCl. Heparin (1 mg/ml) had the same eluting capacity as 0.4 M NaCl, whereas other glycosaminoglycans were only marginally effective. We conclude that the molecular forms of AChE in these vertebrate species interact with heparin, at salt concentrations that are characteristic for asymmetric and globular forms. Within the A and G molecular form groups, no differences were found in the behavior of the different fractions or subtypes, provided that the enzyme samples were free of interfering molecules. If heparin affinity reflects the ability of AChE forms to interact with extracellular matrix components, not only asymmetric but also some globular enzyme forms could be bound to basal laminae under physiological ionic strength conditions.
Murine Neuroblastoma Cells Express Ganglioside Binding Sites on Their Cell Surface
Authors: Fueshko Susan M. , Schengrund Cara‐Lynne
Published: October 2006

Abstract: The ability of S20Y cholinergic, and N115 adrenergic, murine neuroblastoma cells to adhere to immobilized gangliosides was studied. Viable S20Y cells adhered more strongly to GM1‐coated plastic wells than to those coated with GM2, GD1a, or GT1b. The oligosaccharide portion of GM1 inhibited adherence of S20Y cells to GM1‐coated wells, indicating that the carbohydrate moiety of GM1 bore the recognition site. Analysis of S20Y cell adherence to wells coated with derivatives of GM1 indicated that the cells did not adhere to asialo‐GM1 and adherence to the methyl ester or de‐N‐acetyl derivatives was significantly reduced. Expression of the GM1 binding sites by S20Y cells appears to be density dependent; cells harvested at the confluent stage of growth were more adherent than those harvested at the preconfluent stage. Trypsin treatment of the S20Y and N115 cells resulted in a loss of binding to GM1‐coated wells, suggesting that the cell surface GM1 binding site is a protein. In contrast, N115 cells showed no significant difference in their adherence to wells coated with GM1, GD1a, GT1b, Gal‐Cer, asialo‐GM1, or the methyl ester of GM1 when assayed under the same conditions as those imposed on the S20Y cells. The N115 cells did show a reduction in adherence to GM2‐coated wells, suggesting that they recognized the terminal galactosyl moiety.