bluuebunny/crossref_metadata_2025 huggingface dataset:
Glutamate Inhibits Adenylate Cyclase Activity in Dispersed Rat Hippocampal Cells Directly via an N‐Methyl‐d‐Aspartate‐Like Metabotropic Receptor
Authors: Itano Yasuhiro , Murayama Toshihiko , Kitamura Yoshihisa , Nomura Yasuyuki
Published: October 2006

Abstract: Three major subtypes of glutamate receptors that are coupled to cation channels—N‐methyl‐d‐aspartate (NMDA), kainate, and α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionate (AMPA) receptors—are known as ionotropic receptors in the mammalian CNS. Recently, an additional subtype that is coupled to GTP binding proteins and stimulates (or inhibits) metabolism of phosphoinositides has been proposed as a metabotropic receptor. Incubation of dispersed hippocampal cells from adult rats with glutamate or NMDA decreased forskolin‐stimulated cyclic AMP (cAMP) accumulation; half‐maximal effects were obtained with 5.6 ± 2.2 and 6.4 ± 2.3 μM, respectively. Kainate and quisqualate were less potent. The effect of glutamate was antagonized by 2,3‐diaminopropionate and 2‐amino‐5‐phosphonovalerate, NMDA/glutamate receptor antagonists, but not by 0.5 μM Joro spider toxin, a specific blocker of the AMPA receptor. The inhibitory effect of glutamate on cAMP formation was not blocked by 2 μM tetrodotoxin or by the absence of Ca2+. In hippocampal membranes, glutamate, similar to carbachol, inhibited adenylate cyclase activity in a GTP‐dependent manner. These findings suggest that the glutamate inhibition of adenylate cyclase is direct and is not due to a result of the release of other neurotransmitters. The effect of glutamate on cAMP accumulation was observed in an assay medium containing 0.7 mM MgCl2, which is known to inhibit both ionotropic NMDA receptor/channels in the hippocampus and metabotropic NMDA receptors in the cerebellum. The inhibitory effect of glutamate was abolished by pertussis toxin treatment. In conclusion, the rat hippocampus appears to contain a novel class of metabotropic receptors that prefer glutamate and NMDA and is coupled with adenylate cyclase in an inhibitory manner via pertussis toxin‐sensitive GTP binding proteins.
Interactions Between Phospholipase C‐Coupled and N‐Methyl‐D‐Aspartate Receptors in Cultured Cerebellar Granule Cells: Protein Kinase C Mediated Inhibition of N‐Methyl‐D‐Aspartate Responses
Authors: Courtney Michael J. , Nicholls David G.
Published: October 2006

Abstract: The N‐methyl‐D‐aspartate (NMDA) receptor of rat cerebellar granule cells in primary culture is inhibited by phospholipase C‐coupled receptor activation. In the absence of ionotropic agonist, cells modulate their cytoplasmic free Ca2+, [Ca2+]c, in response to stimulation of M3 muscarinic receptors, metabotropic glutamate receptors, and endothelin receptors by the respective agonists carbachol, trans‐l‐amino‐l,3‐cyclopentanedicarboxylic acid, and endothelin‐1. The response is consistent with the ability of phospholipase C‐coupled receptors to release a pool of intracellular Ca2+ and induce a subsequent Ca2+ entry into the cell; both of these responses can be abolished by discharge of internal Ca2+ stores with low concentrations of ionomycin or thapsigargin. In the case of cells stimulated with NMDA, the [Ca2+]c response to the phospholipase C‐coupled agonists is complex and agonist dependent; however, in the presence of ionomycin each agonist produces a partial inhibition of the NMDA component of the [Ca2+]c signal. This inhibition can be mimicked by the protein kinase C activator 4β‐phorbol 12,13‐dibutyrate. It is concluded that NMDA receptors on cerebellar granule cells are inhibited by phospholipase C‐coupled muscarinic M3, glutamatergic, and endothelin receptors via activation of protein kinase C.
Is the Vulnerability of Neurons in the Substantia Nigra of Patients with Parkinson's Disease Related to Their Neuromelanin Content?
Authors: Kastner A. , Hirsch E. C. , Lejeune O. , Javoy‐Agid F. , Rascol O. , Agid Y.
Published: October 2006

Abstract: The contribution of neuromelanin (NM) to the pathogenesis of Parkinson's disease (PD) has long been suspected. In particular, a correlation has been reported between the estimated cell loss in the mesencephalic dopaminergic cell groups and the percentage of NM‐pigmented neurons in these cell groups. To test whether the amount of pigment per cell is a critical factor or whether the presence of NM within a neuron is sufficient to account for the degeneration of dopaminergic neurons, the NM content was measured in each neuron from representative sections throughout the ventral mesencephalon of four control subjects and four patients with PD. Intraneuronal NM was quantified by a densitometric method, using known amounts of synthetic melanin as standards. In control brains, the distribution of melanized neurons in the nigral complex showed a high proportion of lightly melanized neurons in the ventral tegmental area and the pars α and γ of the substantia nigra (SN), whereas heavily melanized neurons were mostly located in the pars β and lateralis of the SN. An inverse relationship was observed between the percentage of surviving neurons in PD compared with controls and the amount of NM they contain, suggesting that the vulnerability of the dopaminergic neurons is related to their NM content. Factors other than NM may be involved in the differential vulnerability of catecholaminergic neurons in PD. In particular, the constant topography of the cell loss suggests that cell position within the nigral complex is a key factor.
pp42/44MAP Kinase Is a Component of the Neurogenic Pathway Utilized by Nerve Growth Factor in PC12 Cells
Authors: Lloyd Elizabeth D. , Wooten Marie W.
Published: October 2006

Abstract: Nerve growth factor‐stimulated mitogen‐activated protein kinase (pp42/44MAP) kinase was characterized by sequential column chromatography on DEAE‐Sephacel, phenyl‐Sepharose CL4B, and S‐200. The kinase displayed an apparent molecular mass of 42 kDa and reacted with an antiphosphotyrosine antibody. Peptide mapping of myelin basic protein revealed the presence of one phosphopeptide that was phosphorylated on Thr‐97. pp42/44MAP kinase activity was dependent on Mg2+ and inhibited by K252a both in vitro and in vivo. Nerve growth factor‐stimulated kinase activation was diminished by down‐regulation of protein kinase C with 200 nM 12‐phorbol 13‐myristate acetate or with staurosporine (1 nM), a protein kinase C inhibitor. Genistein, a protein tyrosine kinase inhibitor, blocked nerve growth factor‐mediated neurite extension as well as diminished activation of pp42/44MAP kinase. Our data demonstrate that activation of this kinase system by nerve growth factor displays a requirement for both protein kinase C as well as protein tyrosine kinase. In addition, other agents that are capable of promoting neurite outgrowth in PC12 cells, such as fibroblast growth factor or dibutryl cyclic AMP, do so independently of activating this kinase system.
Two Pathways of Cyclic GMP Production Through Glutamate Receptor‐Mediated Nitric Oxide Synthesis
Authors: Okada Daisuke
Published: October 2006

Abstract: The selective agonists for the metabotropic glutamate receptor and the ionotropic non‐Ar‐methyl‐D‐aspar‐tate (NMDA) glutamate receptor, (±)‐l‐aminocyclopen‐tane‐/ra/w‐l,3‐dicarboxylic acid (ACPD) and (R,S)‐α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionate (AMPA), respectively, increased the cyclic GMP (cGMP) content in cerebellar slices prepared from adult rats. The ACPD‐induced rise in cGMP level was blocked by compounds known to antagonize metabotropic glutamate receptors, such as DL‐2‐amino‐3‐phosphonopropionic acid and L‐2‐amino‐4‐phosphonobutyric acid, but not by ionotropic glutamate receptor antagonists, D‐2‐amino‐5‐phosphonovale‐ric acid and 6 ‐ cyano ‐ 7 ‐ nitroquinoxaline ‐2,3– dione (CNQX), whereas the AMPA‐induced rise in cGMP level was suppressed by CNQX. Both rises in cGMP level involved nitric oxide synthase (NOS), because NG‐methyl‐L‐arginine (NMLA), an inhibitor of NOS, blocked both cGMP level rises, and excess L‐arginine reversed the effect of NMLA. After lithium chloride treatment, which could exhaust phosphatidylinositol phosphates, ACPD no longer increased cGMP levels, whereas AMPA was still effective. In a calcium‐free medium, ACPD still induced a rise in cGMP level, whereas AMPA did not. When the molecular layer was isolated to determine the cGMP content separately from that in the rest of the cerebellar cortex, it was found that ACPD raised the cGMP level mainly in the molecular layer, whereas AMPA raised it in both sections. These results suggest that ACPD enhances the cGMP level through activation of NOS independently of extracellular calcium, most likely by calcium release from intracellular stores triggered by metabotropic glutamate receptors linked to phosphoinositide breakdown, whereas AMPA activates the enzyme by calcium entry from the extracellular space triggered by ionotropic non‐NMDA glutamate receptors.
Altered Microviscosity at Brain Membrane Surface Induces Distinct and Reversible Inhibition of Opioid Receptor Binding
Authors: Lazar Dan F. , Medzihradsky Fedor
Published: October 2006

Abstract: In synaptosomal membranes from rat and monkey brain cortex, the addition of petroselenic (18:1, cis‐Δ6) acid, oleic (18:1, m‐Δ9) acid, and vaccenic (18:1, cis‐Δ11) acid or their corresponding methyl esters at 0.5 μmol/mg of membrane protein caused a similar 7–10% decrease in the microviscosity of the membrane core, whereas at the membrane surface the microviscosity was reduced 5–7% by the fatty acids but only 1% by their methyl esters. Concomi‐tantly, the fatty acids, but not the methyl esters, inhibited the specific binding of the tritiated μ‐, δ‐, and K‐opioids Tyr‐D‐Ala‐Gly‐(Me)Phe‐Gly‐ol (DAMGO), [D‐Pen2,D‐Pen5]‐enkephalin (DPDPE), and U69,593, respectively. As shown with oleic acid, the sensitivity of opioid receptor binding toward inhibition by fatty acids was in the order δ > μκk, whereby the binding of [3H]DPDPE was abolished, but significant inhibition of [3H]U69,593 binding, determined in membranes from monkey brain, required membrane modification with a twofold higher fatty acid concentration. Except for the unchanged KD of [3H]U69,593, the inhibition by oleic acid involved both the Bmax and affinity of opioid binding. Cholesteryl hemisuccinate (0.5–3 μmol/mg of protein), added to membranes previously modified by fatty acids, reversed the fluidization caused by the latter compounds and restored inhibited μ‐, δ‐, and k‐opioid binding toward control values. In particular, the Bmax of [3H]‐DPDPE binding completely recovered after being undetectable. The results implicate membrane surface fluidity in the modulation of opioid receptor binding, reveal distinct sensitivity of δ, μ, and K receptors toward that modulation, and identify unsaturated fatty acids and cholesterol as possible endogenous regulators of opioid receptor function.
Distribution of Calcitonin Gene‐Related Peptide in Rat and Rabbit Spinal Cords: Effect of Intrathecal 5,7‐Dihydroxytryptamine
Authors: Fone K. C. F.
Published: October 2006

Abstract: Calcitonin gene‐related peptide‐like immunoreactivity (CGRP‐LI) was measured in selected regions of the cervical, thoracic, and lumbar spinal cord of untreated rabbits and, following intrathecal injection of the serotonergic neurotoxin 5,7‐dihydroxytryptamine (5,7‐DHT), in the thoracolumbar cord in rats using a sheep antiserum raised against tyrosine0 calcitonin gene‐related peptide28‐37. In the cervical, thoracic, and lumbar segments of the rabbit spinal cord, CGRP‐LI levels were 15–50‐fold higher in the dorsal than in the ventral grey region in the same segment. The only segmental variation in CGRP‐LI levels was in the dorsal white region, where levels in the thoracic cord were lower than those in cervical or lumbar segments. Within individual spinal segments, the pattern of distribution of CGRP‐LI in the rabbit spinal cord was analogous to that in other species previously examined, including rat, human, and cat spinal cord. Intrathecal injection of 5,7‐DHT, which caused 85–91% depletion of 5‐hydroxytryptamine and 5‐hydroxyindoleacetic acid from the thoracolumbar ventral spinal cord, did not affect choline acetyltransferase activity, which is colocalized with CGRP in motoneurones in this spinal cord region. In contrast, intrathecal 5,7‐DHT produced a threefold increase in CGRP‐LI in the ventral thoracolumbar cord, suggesting that spinal motoneurones selectively increase production of CGRP 10 days after neurotoxin‐induced denervation of bulbospinal raphe neuronal input.
Lesion‐Induced Changes in the Production of Newly Synthesized and Secreted Apo‐E and Other Molecules Are Independent of the Concomitant Recruitment of Blood‐Borne Macrophages into Injured Peripheral Nerves
Authors: Aamar S. , Saada A. , Rotshenker S.
Published: October 2006

Abstract: Peripheral nerve injury produces Wallerian degeneration characterized by a change in the composition of resident nonneuronal cells: macrophages are recruited from the circulation to join Schwann, fibroblast, and endothelial cells. At the same time, the nonneuronal cell population exhibits, as a whole, alterations in synthesis and secretion of diffusible molecules, some of which are instrumental in nerve repair mechanisms. In this study, we determined whether changes in the production of secreted molecules depend on the concomitant modification in cell composition. Therefore, we studied the secretion of newly synthesized molecules by defined cell populations of intact nerves, intact nerve explants undergoing in vitro axonal degeneration, in vivo degenerating nerves, and recruited cells. Nerves were incubated in serum‐free, [35S]methionine‐containing media. Secreted, radioactively labeled proteins were precipitated from the medium and analyzed by gel electrophoresis. Reduced production of 43‐, 46‐, and 48‐kDa proteins and increased production of 33–34‐, 37‐, 49‐, 59–, and 67‐kDa proteins were detected in in situ degenerating nerves. High‐density ultracentrifugation and immunoblot analysis revealed that the 33–34‐kDa protein is apolipoprotein‐E (apo‐E). Similar alterations in the production of these molecules were detected in intact nerve explants from which blood‐borne cells were excluded. Apo‐E, 37‐, 49‐, 59‐, and 67‐kDa proteins were also produced in frozen nerves that lacked the intact nerve nonneuronal cell population. Instead, these preparations contained blood‐borne cells, primarily macrophages. Thus, change in the production of a substantial number of secreted molecules, apo‐E included, is a characteristic response to axonal disintegration of the nonneuronal cells resident in intact nerves. Recruited macrophages, although not required, contribute to the production of apo‐E and other secreted molecules. The production of apo‐E and 45‐kDa proteins was inhibited, and that of 37‐kDa proteins increased in the presence of NH4Cl, further suggesting that lysosomal activity plays a role in the regulation of the production of these molecules.
Tetanus Toxin Inhibits Depolarization‐Stimulated Protein Phosphorylation in Rat Cortical Synaptosomes: Effect on Synapsin I Phosphorylation and Translocation
Authors: Presek Peter , Jessen Sönke , Dreyer Florian , Jarvie Paula E. , Findik Duygu , Dunkley Peter R.
Published: October 2006

Abstract: Synapsin I, a prominent phosphoprotein in nerve terminals, is proposed to modulate exocytosis by interaction with the cytoplasmic surface of small synaptic vesicles and cytoskeletal elements in a phosphorylation‐dependent manner. Tetanus toxin (TeTx), a potent inhibitor of neurotransmitter release, attenuated the depolarization‐stimulated increase in synapsin I phosphorylation in rat cortical particles and in synaptosomes. TeTx also markedly decreased the translocation of synapsin I from the small synaptic vesicles and the cytoskeleton into the cytosol, on depolarization of synaptosomes. The effect of TeTx on synapsin I phosphorylation was both time and TeTx concentration dependent and required active toxin. One‐ and two‐dimensional peptide maps of synapsin I with V8 proteinase and trypsin, respectively, showed no differences in the relative phosphorylation of peptides for the control and TeTx‐treated synaptosomes, suggesting that both the calmodulin‐and the cyclic AMP‐dependent kinases that label this protein are equally affected. Phosphorylation of synapsin IIb and the B‐50 protein (GAP43), a known substrate of protein kinase C, was also inhibited by TeTx. TeTx affected only a limited number of phosphoproteins and the calcium‐dependent decrease in dephosphin phosphorylation remained unaffected. In vitro phosphorylation of proteins in lysed synaptosomes was not influenced by prior TeTx treatment of the intact synaptosomes or by the addition of TeTx to lysates, suggesting that the effect of TeTx on protein phosphorylation was indirect. Our data demonstrate that TeTx inhibits neurotransmitter release, the phosphorylation of a select group of phosphoproteins in nerve terminals, and the translocation of synapsin I. These findings contribute to our understanding of the basic mechanism of TeTx action.
Nuclear Magnetic Resonance Studies on the Effects of Decreased External Sodium on Guinea Pig Cerebral Cortex Slices and the Permeabilities of Various Sodium Substitutes
Authors: Brooks K. J. , Bachelard H. S.
Published: October 2006

Abstract: Decreasing the external sodium concentration ([Na+]e) to 10 mM in the presence of 280 mM sucrose had no significant effect on phosphocreatine (PCr) or on intracellular pH (pHi) as assessed using 31P nuclear magnetic resonance spectroscopy. Zero [Na+]e in the presence of 300 mM sucrose caused a fall in PCr levels to 50% of control values, and the pHi fell to 6.85 from a control value of 7.30. 1H nuclear magnetic resonance spectroscopy confirmed that the sucrose had not entered the tissue. The decreases in PCr content and in pHi, known to occur on depolarization using 40 mM external potassium concentration ([K+]c), were further decreased in the presence of 10 mM [Na+]e, to 51.4 ± 4.0 and 6.80 ± 0.10% of control values, respectively. The free intracellular magnesium concentration was significantly increased from a control value of 0.37 ± 0.10 mM to 0.66 ±0.13 mM (p < 0.001), when [Na+]e was decreased to 10 mM, but was not further affected by high [K+]e or zero Na+. Membrane permeabilities of the sodium substitutes N‐methyl‐D‐glucamine (NMG), tris(hydroxymethyl)aminomethane (Tris), tetramethylammonium (TMA), and choline were assessed using 1H nuclear magnetic resonance spectroscopy. In the presence of 10 mM [Na+]e, NMG, TMA, and choline (all at 140 mM) were taken up and remained within the tissue for at least 2 h, but no uptake of Tris (140 mM) or sucrose (above) could be detected. Tissue lactate levels (from the lactate/N‐acetyl aspartate ratio) increased in the presence of the substitutes that were taken up, although no change in pH was detected.
The Adenosine Analogue N6‐L‐Phenylisopropyladenosine Inhibits Catecholamine Secretion from Bovine Adrenal Medulla Cells by Inhibiting Calcium Influx
Authors: Chern Yi‐Juang , Bott Marga , Chu Po‐Ju , Lin Yi‐Jen , Kao Lung‐Sen , Westhead Edward W.
Published: October 2006

Abstract: We reported earlier that adenine nucleotides and adenosine inhibit acetylcholine‐induced catecholamine secretion from bovine adrenal medulla chromaffin cells. In this article, we used an adenosine analogue, N6‐L‐phenyliso‐propyladenosine (PIA), to study the mechanism underlying inhibition of catecholamine secretion by adenosine. PIA inhibits secretion induced by a nicotinic agonist, 1,1‐di‐methyl‐4‐phenylpiperazinium, or by elevated external K+. The half‐maximal effect on 1,1‐dimethyl‐4‐phenylpiperazinium‐induced secretion occurred at ∼5 ± 10‐‐5M. The inhibition is immediate and reversible. Fura‐2 measurements of cytosolic free Ca2+ indicate that PIA inhibits Ca2+ elevation caused by stimulation; measurements of 45Ca2+ influx show that PIA inhibits uptake of Ca2+. PIA does not inhibit calcium‐evoked secretion from digitonin‐permeabilized cells, nor does PIA cause any significant change in the dependence of catecholamine secretion on calcium concentration. These data suggest that inhibition by PIA occurs at the level of the voltage‐sensitive calcium channel.
Brain Protein Synthesis in the Conscious Rat Using L‐[35S]Methionine: Relationship of Methionine Specific Activity Between Plasma and Precursor Compartment and Evaluation of Methionine Metabolic Pathways
Authors: Grange Eric , Gharib Abdallah , Lepetit Patrick , Guillaud Josette , Sarda Nicole , Bobillier Pierre
Published: October 2006

Abstract: The method previously developed for the measurement of rates of methionine incorporation into brain proteins assumed that methionine derived from protein degradation did not recycle into the precursor pool for protein synthesis and that the metabolism of methionine via the transmethylation pathway was negligible. To evaluate the degree of recycling, we have compared, under steady‐state conditions, the specific activity of L‐[35S]methionine in the tRNA‐bound pool to that of plasma. The relative contribution of methionine from protein degradation to the precursor pool was 26%. Under the same conditions, the relative rate of methionine flux into the transmethylation cycle was estimated to be 10% of the rate of methionine incorporation into brain proteins. These results indicate the following: (a) there is significant recycling of unlabeled methionine derived from protein degradation in brain; and (b) the metabolism of methionine is directed mainly towards protein synthesis. At normal plasma amino acid levels, methionine is the amino acid which, to date, presents the lowest degree of dilution in the precursor pool for protein synthesis. L‐[35S]‐Methionine, therefore, presents radiobiochemical properties required to measure, with minimal underestimation, rates of brain protein synthesis in vivo.
Melatonin Increases Serotonin N‐Acetyltransferase Activity and Decreases Dopamine Synthesis in Light‐Exposed Chick Retina: In Vivo Evidence Supporting Melatonin‐Dopamine Interaction in Retina
Authors: Nowak Jerzy Z. , Kazula Arkadiusz , Gotembiowska Krystyna
Published: October 2006

Abstract: The administration of melatonin, either peripherally (0.01–10 mg/kg) or intraocularly (0.001–10 μmol/eye), to light‐exposed chicks dose‐dependently increased serotonin N‐acetyltransferase (NAT) activity in retina but not in pineal gland. The effect of melatonin was slightly but significantly reduced by luzindole (2‐benzyl‐N‐acetyltryptamine), and not affected by two other purported melatonin antagonists, N‐acetyltryptamine and N‐(2,4‐dinitrophenyl)‐5‐methoxytryptamine (ML‐23). The elevation of the enzyme activity induced by melatonin was substantially stronger than that evoked by 5‐hydroxytryptamine, N‐acetyl‐5‐hydroxytryptamine, or 5‐methoxytryptamine. The melatonin‐evoked rise in the retinal NAT activity was counteracted by two dopamine D2 receptor agonists, quinpirole and apomorphine, and prevented by the dopamine D2 receptor blocker spiroperidol, and by an inhibitor of dopamine synthesis, α‐methyl‐p‐tyrosine. Melatonin (0.1–10 mgJ.kg i.p.) dose‐dependently decreased the levels of dopamine and 3,4‐dihydroxyphenylacetic acid (DOPAC), as well as the DOPACJ.dopamine ratio, in chick retina but not in forebrain. The results obtained (1) indicate that melatonin in vivo potently inhibits dopamine synthesis selectively in retina, and (2) suggest that the increase in retinal NAT activity evoked by melatonin in light‐exposed chicks is an indirect action of the compound, and results from the disinhibition of the NAT induction process from the dopaminergic (inhibitory) signal. The results provide in vivo evidence supporting the idea (derived on the basis of in vitro findings) that a mutually antagonistic interaction between melatonin and dopamine operates in retinas of living animals.
Eclosion Hormone Stimulates Cyclic GMP Levels in Manduca sexta Nervous Tissue via Arachidonic Acid Metabolism with Little or No Contribution from the Production of Nitric Oxide
Authors: Morton David B. , Giunta Michael A.
Published: October 2006

Abstract: The neuropeptide eclosion hormone acts directly on the nervous system of the tobacco hornworm, Manduca sexta, to trigger ecdysis behavior at the end of each molt. Previous studies have shown that the action of eclosion hormone is mediated via the intracellular messenger cyclic GMP. In the present study we have investigated the mechanisms involved in the eclosion hormone‐stimulated increases in cyclic GMP. No stimulation of guanylate cyclase was seen in homogenized nervous tissue, suggesting that eclosion hormone does not directly stimulate a membranebound form of guanylate cyclase. Nitric oxide synthase inhibitors, N‐methylarginine and nitroarginine, had no effect on eclosion hormone‐stimulated cyclic GMP levels. By contrast, 4‐bromophenacyl bromide, an inhibitor of arachidonic acid release, and nordihydroguaiaretic acid, an inhibitor of arachidonic acid metabolism, almost completely abolished the eclosion hormone‐stimulated cyclic GMP increase. We hypothesize that eclosion hormone receptors are coupled to a lipase, activation of which causes the release of arachidonic acid. Either the arachidonic acid directly stimulates the soluble guanylate cyclase or further metabolism of arachidonic acid yields compounds that activate guanylate cyclase.
Biochemical and Autoradiographical Determination of Protein Synthesis in Experimental Brain Tumors of Rats
Authors: Widmann R. , Kocher M. , Ernestus R.‐I. , Hossmann K.‐A.
Published: October 2006

The rate of leucine incorporation into brain proteins was studied in rats with experimental brain tumors produced by intracerebral transplantation of the glioma clone F98. Incorporation was measured with [14C]leucine using a controlled infusion technique for maintaining constant specific activity of [14C]leucine in plasma, followed by quantitative autoradiography and biochemical tissue analysis. After 45 min the specific activity of free [14C]leucine in plasma was 2.5–3 times higher than in brain and brain tumor, indicating that the precursor pool for protein synthesis was fueled both by exogenous (plasma‐derived) and endogenous (proteolysis‐derived) amino acids. Endogenous recycling of amino acids amounted to 73% of total free leucine pool in brain tumors and to 60–70% in normal brain. Taking endogenous amino acid recycling into account, leucine incorporation was 78.7 ± 16.0 nmol/g of tissue/min in brain tumor, and 17.2 ± 4.2 and 9.7 ± 3.3 nmol/g/min in normal frontal cortex and striatum, respectively. Leucine incorporation within tumor tissue was markedly heterogeneous, depending on the local pattern of tumor proliferation and necrosis. Our results demonstrate that quantitative measurement of leucine incorporation into brain proteins requires estimation of recycling of amino acids derived from proteolysis and, in consequence, biochemical determination of the free amino acid precursor pool in tissue samples. With the present approach such measurements are possible and provide the quantitative basis for the evaluation of therapeutic interventions.
Specific Expression of N‐Acetylaspartate in Neurons, Oligodendrocyte‐Type‐2 Astrocyte Progenitors, and Immature Oligodendrocytes In Vitro
Authors: Urenjak Jutta , Williams Stephen R. , Gadian David G. , Noble Mark
Published: October 2006

To test the specificity of N‐acetylaspartate (NAA) as a neuronal marker for proton nuclear magnetic resonance (1H NMR) spectroscopy, purified and characterized cultured cells were analyzed for their NAA content using both 1H NMR and HPLC. Cell types studied included cerebellar granule neurons, type‐1 astrocytes, meningeal cells, oligodendrocyte‐type‐2 astrocyte (O–2A) progenitor cells, and oligodendrocytes. A high concentration of NAA was found in extracts of cerebellar granule neurons (approximately 12 nmol/mg of protein), whereas NAA remained undetectable in purified type‐1 astrocytes, meningeal cells, and mature oligodendrocytes. However, twice the neuronal level of NAA was found in O‐2A progenitors grown in vitro. In addition significant levels of NAA were also detected in cultures of immature oligodendrocytes. Our data partly support previous suggestions that NAA may be a useful neuronal marker for 1H NMR spectroscopic examination of the adult brain. However, they also raise the further possibility that alterations of NAA associated with some specific brain disorders, particularly disorders seen in newborn and young children, may reflect abnormalities in the development of oligodendroglia or their precursors.
Reduced Neuropeptide Y Concentrations in Suicide Brain
Authors: Widdowson Peter S. , Ordway Gregory A. , Halaris Angelos E.
Published: October 2006

Neuropeptide Y (NPY) was measured in postmortem brain tissue from victims of suicide and from individuals dying a sudden natural or accidental death (controls). Concentrations of NPY‐immunoreactivity were measured by radioimmunoassay in frontal cortex (BA 10), temporal cortex (BA 22), caudate nucleus, and cerebellum. Concentrations of NPY‐immunoreactivity were significantly lower in postmortem frontal cortex (−14%) and caudate nucleus (−27%) from suicide victims compared with age‐matched controls. A subgroup of suicides with evidence of a history of depression revealed more robust reductions in concentrations of NPY‐immunoreactivity in frontal cortex and caudate nucleus, as did four individuals who died from natural causes and also were described as having a possible history of depression. Concentrations of NPY‐immunoreactivity in temporal cortex and cerebellum from victims of suicide or from the subgroup of subjects with a possible history of depression were not significantly different from those of age‐matched controls. We suggest there is a deficit in the brain NPY system leading to region‐specific reductions in peptide concentrations in subjects who have a history of depression.
Glutamate, of the Endogenous Primary α‐Amino Acids, Is Specifically Released from Hair Cells by Elevated Extracellular Potassium
Authors: Drescher Marian J. , Drescher Dennis G.
Published: October 2006

A hair cell (octavolateralis mechanoreceptor cell) sheet preparation from the trout saccular macula was superfused with bicarbonate‐based physiological saline. Among the primary amine‐containing compounds resolved by cation‐exchange HPLC, glutamate alone was released in a statistically significant manner with elevation of extracellular [K+] from 3.5 to 14 mM in the presence of 1.8 mM calcium. Release of glutamate averaged 10.9 ± 2.5 pmol (mean ± SEM) over a 10‐min period for a hair cell sheet preparation representing 20 μg of cell protein. No potassium‐evoked release of glutamate was observed in 0 mM calcium/ 10 mM magnesium saline, suggesting calcium dependency. Because the sheet preparation, by the method of its isolation, contained only the hair cell as the intact cell type, release of glutamate, induced by relatively small increases in extracellular potassium, can be attributed directly to the receptor cell. The specific release of glutamate and its block by magnesium are consistent with the hypothesis that glutamate is one neurotransmitter/neuromodulator mediating receptoneural transmission in the octavolateralis periphery.
Identification of an Activator of the Microtubule‐Associated Protein 2 Kinases ERK1 and ERK2 in PC12 Cells Stimulated with Nerve Growth Factor or Bradykinin
Authors: Ahn Natalie G. , Robbins David J. , Haycock John W. , Seger Rony , Cobb Melanie H. , Krebs Edwin G.
Published: October 2006

Treatment of PC12 pheochromocytoma cells with nerve growth factor (NGF) or bradykinin leads to the activation of extracellular signal‐regulated kinases ERK1 and ERK2, two isozymes of microtubule‐associated protein 2 (MAP) kinase that are present in numerous cell lines and regulated by diverse extracellular signals. The activation of MAP kinase is associated with its phosphorylation on tyro‐sine and threonine residues, both of which are required for activity. In the present studies, we have identified a factor in extracts of PC12 cells treated with NGF or bradykinin, named MAP kinase activator, that, when reconstituted with inactive MAP kinase from untreated cells, dramatically increased MAP kinase activity. Activation of MAP kinase in vitro by this factor required MgATP and was associated with the phosphorylation of a 42‐ (ERK1) and 44‐kDa (ERK2) polypeptide. Incorporation of 32P into ERK1 and ERK2 occurred primarily on tyrosine and threonine residues and was associated with a single tryptic peptide, which is identical to one whose phosphorylation is increased by treatment of intact PC12 cells with NGF. Thus, the MAP kinase activator identified in PC12 cells is likely to be a physiologically important intermediate in the signaling pathways activated by NGF and bradykinin. Moreover, stimulation of the activator by NGF and bradykinin suggests that tyrosine kinase receptors and guanine nucleotide‐binding protein‐coupled receptors are both capable of regulating these pathways.
Ethanol‐Induced Increase in Endogenous Dopamine Release May Involve Endogenous Opiates
Authors: Widdowson Peter S. , Holman R. Bruce
Published: October 2006

The effect of opiate peptides on basal and potassium‐stimulated endogenous dopamine (DA) release from striatal slices was studied in vitro. Dual stimulation of the striatal slices gave a reproducible increase in DA release that was calcium dependent. Addition of the δ‐opiate receptor agonists Met5‐enkephalin, [D‐Ala2,D‐Leu5]enkephalin (DADLE), and [D‐Ser]Leu‐enkephalin‐Thr (DSLET), increased the basal DA release without affecting potassium‐stimulated release in a dose‐dependent manner. The effect of DADLE was antagonized by the addition of naloxone. In contrast, the μ‐opioid receptor agonist [D‐Ala2, N‐MePhe4,Gly‐ol5]enkephalin (DAGO) and the e‐opioid agonist β‐endorphin inhibited the stimulated DA release without changing the basal release. The inhibitory effect of DAGO on potassium‐stimulated release was antagonized by naloxone. The addition of ethanol (75 mM) to the incubation media produced a delayed increase of both the basal and stimulated DA release. There was no change in stimulated DA release when the change in basal release was subtracted, suggesting that ethanol produced a dose‐dependent, selective increase in basal DA release. Naloxone and the selective δ‐opiate antagonist ICI 174864 inhibited the eth‐anol‐induced increase in basal DA release. Naloxone and ICI 174864 added alone did not alter either basal or stimulated DA release. We therefore suggest that the ethanol‐in‐duced increase in basal DA release is an indirect effect involving an endogenous δ‐opiate agonist.
Screening of Thiol Compounds: Depolarization‐Induced Release of Glutathione and Cysteine from Rat Brain Slices
Authors: Zängerle Leo , Cuénod Michel , Winterhalter Kaspar H. , Do Kim Quang
Published: October 2006

Superfusates from rat brain slices were screened for thiol compounds after derivatization with monobromo‐bimane by reversed‐phase HPLC. Only glutathione and cysteine were detected. The Ca2+‐dependent release of these compounds from slices of different regions of rat brain was investigated, applying a highly sensitive and reproducible quantification method, based on reduction of superfusates with dithiothreitol, reaction of thiols with iodoacetic acid, precolumn derivatization with o‐phthalaldehyde reagent solution, and analysis with reversed‐phase HPLC. This methodology allowed determination of reduced and total thiols in aliquots of the same superfusates. Mostly reduced glutathione and cysteine were released upon K+ depolarization and the Ca2+ dependency suggests that they originate from a neuronal compartment. The GSH release was most prominent in the mesodiencephalon, cortex, hippocampus, and striatum and lowest in the pons‐medulla and cerebellum. This underscores a physiologically significant role for glutathione in CNS neurotransmission.
In Vivo Evidence that Lithium Inactivates Gi Modulation of Adenylate Cyclase in Brain
Authors: Masana Monica I. , Bitran Jose A. , Hsiao John K. , Potter William Z.
Published: October 2006

In vivo microdialysis of cyclic AMP from prefron‐tal cortex complemented by ex vivo measures was used to investigate the possibility that lithium produces functional changes in G proteins that could account for its effects on adenylate cyclase activity. Four weeks of lithium administration (serum lithium concentration of 0.85 ±0.05 mM; n= 11) significantly increased the basal cyclic AMP content in dialysate from prefrontal cortex of anesthetized rats. Forskolin infused through the probe increased dialysate cyclic AMP, but the magnitude of this increase was unaffected by chronic lithium administration. Inactivation of the inhibitory guanine nucleotide binding protein Gi with pertussis toxin increased dialysate cyclic AMP in control rats, as did stimulation with cholera toxin (which activates the stimulatory guanine nucleotide binding protein Gs). The effect of pertussis toxin was abolished following chronic lithium, whereas the increase in cyclic AMP after cholera toxin was enhanced. In vitro pertussis toxin‐catalyzed ADP ribosyla‐tion of αi (and αo) was increased by 20% in prefrontal cortex from lithium‐treated rats, but the αi and αs contents (as determined by immunoblot) as well as the cholera toxin‐catalyzed ADP ribosylation of αs were unchanged. Taken together, these results suggest that chronic lithium administration may interfere with the dissociation of Gi into its active components and thereby remove a tonic inhibitory influence on adenylate cyclase, with resultant enhanced basal and cholera toxin‐stimulated adenylate cyclase activity.
Cerebral Acid Buffering Capacity at Different Ages Measured In Vivo by 31P and 1H Nuclear Magnetic Resonance Spectroscopy
Authors: Corbett Ronald J. T. , Laptook Abbot R. , Garcia Damian , Ruley Joan I.
Published: October 2006

Cerebral acidosis occurring during ischemia has been proposed as one determinant of tissue damage. Newborn animals appear to be less susceptible to ischemic tissue damage than adults. One possible component of ischemic tolerance could derive from maturational differences in the extent of acid production and buffering in newborns compared to adults. The purpose of this study was to measure the dependency of acid production on the blood plasma glucose concentrations and acid buffering capacity of piglets at different stages of development. Complete ischemia was induced in 29 piglets ranging in postconceptual age from 111 to 156 days (normal term conception, 115 days). Brain buffering capacity during the first 30 min of ischemia was quantified in vivo, via 31P and 1H nuclear magnetic resonance (NMR) spectroscopy, by measuring the change in intracellular brain pH for a given change in the concentration of compounds that contribute to the production of hydrogen ions. Animals from all four age groups showed a similar linear correlation between preischemia blood glucose concentration and intracellular pH after 30 min of ischemia. For each animal the slope of the plot of intracellular pH versus cerebral buffer base deficit was used to calculate the buffer capacity. Using data obtained over the entire 30 min of ischemia, there was no difference in the mean buffer capacity of the different age groups, nor was there a significant correlation between buffer capacity and age. However, there was a significant increase in buffer capacity for the intracellular pH range 6.6‐6.0, compared to 7.0‐6.6, for all age groups. No significant differences in buffer capacity for these two pH ranges were observed between any of the age groups. Acid buffering capacity was also measured by performing pH titrations on brain tissue homogenized in the presence of inhibitors of glycolysis and creatine kinase. Plots of homogenate pH versus buffer base deficit showed a nonlinear trend similar to that seen in vivo, indicating an increase in buffer capacity as intracellular pH decreases. A comparison of newborn and 1‐month‐old brain tissue frozen under control conditions or after 45 min of ischemia revealed no differences that could be attributed to age and a slight decrease in buffer capacity of ischemic brain compared to control brain tissue homogenates. There was no difference between the brain buffering capacity measured in vivo using 31P and 1H NMR and that measured in vitro using brain homogenates.
Effect of Intracellular Glutamine on the Uptake of Large Neutral Amino Acids in Astrocytes: Concentrative Na+‐Independent Transport Exhibits Metastability
Authors: Brookes Neville
Published: October 2006

To examine whether the concentration gradient of glutamine (Gln) drives concentrative Na+‐independent uptake of neutral amino acids (NAA) in mouse cerebral astrocytes, uptake was compared in “Gln‐depleted” and “Gin‐replete” cultures. Uptake (30 min in Na+‐free buffer) of histidine, kynurenine, leucine, tyrosine, and a model substrate for System L transport was 70–150% greater in Gin‐replete cultures. Phenylalanine uptake was not affected. All of these NAA trans‐stimulated the export of Gln from astrocytes. However, the increase in NAA uptake was sustained even though the Gln content of Gin‐replete cultures declined. Also, uptake of Gln itself was enhanced in Gln‐re‐plete cultures. Thus, countertransport of Gln was insufficient to explain the enhancement of NAA uptake. Enhanced uptake was restored, and could be magnified, by reloading Gin‐depleted cultures either with Gln or with histidine. It is suggested that substrate‐induced asymmetry and molecular hysteresis in the Na+‐independent carrier could account for the sustained enhancement of NAA uptake. Only histidine and kynurenine were concentrated comparably to Gin (15‐ to 29‐fold at 1 mM in Na+‐free buffer). The other NAA were four to six times less concentrated. At least two Na+‐dependent transport systems also supported the concentration gradient of Gln in regular buffer.
In Vivo Partial Inactivation of Dopamine D1 Receptors Induces Hypersensitivity of Cortical Dopamine‐Sensitive Adenylate Cyclase: Permissive Role of α1‐Adrenergic Receptors
Authors: Trovero Fabrice , Hervé Denis , Blanc Gérard , Glowinski Jacques , Tassin Jean‐Pol
Published: October 2006

As shown by autoradiography, peripheral injections of N‐ethoxycarbonyl‐2‐ethoxy‐1,2‐dihydroquinoline (EEDQ) induced a dose‐dependent decrease of [3H]SCH 23390 and [3H]prazosin high‐affinity binding sites in the rat prefrontal cortex. EEDQ showed similar efficacy in inactivating cortical and striatal dopamine (DA) D1 receptors, whereas prazosin‐sensitive α1‐adrenergic receptors were more sensitive to the action of the alkylating agent, as for all doses of EEDQ tested (from 0.8 to 3 mg/kg, i.p.), the decrease in cortical [3H]SCH 23390 binding was less pronounced than that of [3H]prazosin. The effects of EEDQ on [3H]SCH 23390 binding and DA‐sensitive adenylate cyclase activity were then simultaneously compared in individual rats. In the striatum, whatever the dose of EEDQ used, the decrease of DA‐sensitive adenylate cyclase activity was always lower than that of D1 binding sites, suggesting the occurrence of a large proportion of spare D1 receptors. In the prefrontal cortex, a significant increase in DA‐sensitive adenylate cyclase activity was observed in rats treated with a low dose of EEDQ (0.8 mg/kg), this effect being associated with a slight reduction in [3H]SCH 23390 binding sites (‐20%). Parallel decreases in the enzyme activity and D1 binding sites were observed with higher doses. The EEDQ‐induced supersensitivity of DA‐sensitive adenylate cyclase did not occur in rats in which the decrease in [3H]prazosin binding sites was higher than 35%. Demonstrating further a role of noradrenergic transmission of the α1 type in the regulation of the cortical D1 receptor sensitivity, the blockade of α1‐adrenergic receptors by prazosin prevented the appearance of the EEDQ‐induced supersensitivity of DA‐sensitive adenylate cyclase in groups of rats exhibiting similar decreases in the density of D1 binding sites (‐20%). The DA‐sensitive adenylate cyclase activity was significantly lower in cortices of animals pretreated with prazosin than in those not treated with the α1 antagonist. These results are discussed in relation to previous studies indicating that prazosin pretreatment also abolishes the locomotor hyperactivity induced by bilateral electrolytic lesions of the ventral teg‐mental area.
Inositol Lipids and Signal Transduction in the Nervous System: An Update
Authors: Fisher Stephen K. , Heacock Anne M. , Agranoff Bernard W.
Published: October 2006

Abstract: The role that inositol lipids play in cellular signaling events in eukaryotic cells remains one of the most intensively investigated areas of cell biology. In this respect, phosphoinositide‐mediated signal transduction in the CNS is no exception; major advances have been made since a previous review on this subject (Fisher and Agranoff, 1987). Not only have stimulated phosphoinositide turnover and its physiological sequelae been demonstrated repeatedly in a variety of neural preparations, but, in addition, the detailed molecular mechanisms underlying these events continue to unfold. Here we review the progress that has occurred in selected aspects of this topic since 1987. In the first two sections of this article, emphasis is placed on novel functional roles for the inositol lipids and on recent insights into the molecular characteristics and regulation of three key components of the phosphoinositide signal transduction system, namely, the inositol lipid kinases, phospholipases C (PLCs), and the inositol 1,4,5‐trisphosphate[I(1,4,5)P3] receptor. The metabolic fate of I(1,4,5)P3 in neural tissues, as well as its control, is also detailed. Later we focus on identification of the multiple receptor subtypes that are coupled to inositol lipid turnover and discuss possible strategies for intervention into phosphoinositide‐mediated signal transduction. Due to space limitations, an extensive evaluation of the diacylglycerol/protein kinase C (DAG/PKC) limb of the signal transduction pathway is not included (for reviews, see Nishizuka, 1988; Kanoh et al., 1990).