bluuebunny/crossref_metadata_2025 huggingface dataset:
Biochemical Responses Mediated by N‐Methyl‐d‐Aspartate Receptors in Rat Cortical Slices Are Differentially Sensitive to Magnesium
Authors: Gonzales Rueben A.
Published: October 2006

Abstract: The effects of magnesium on the inhibition of phosphoinositide (PI) hydrolysis and the stimulation of [3H]norepinephrine release by N‐methyl‐d‐aspartate (NMDA) in rat cortical slices were investigated. Removal of the magnesium from the buffer resulted in a small reduction of the inhibitory effect of 100 μM NMDA (34% inhibition in the absence of magnesium, compared with 51% for the control) when slices were coincubated with NMDA and carbachol. Addition of 10 mM Mg2+ also allowed the inhibitory effect of 100 μM NMDA on carbachol‐stimulated PI hydrolysis to be expressed (44% inhibition) under these conditions. Concentration–effect curve analysis for the NMDA‐induced inhibition of carbachol‐stimulated PI hydrolysis indicated that the IC50 for NMDA was decreased from 14.9 μM for the control to 4.2 μM in the absence of magnesium. The absence of magnesium also had small effects on the concentration–effect curve for (+)‐5‐methyl‐10,11‐dihydro‐5H‐dibenzo[a,d]cyclohepten‐5,10‐imine hydrogen maleate reversal of the inhibitory effects of NMDA on carbachol‐stimulated PI hydrolysis. The absence of magnesium also shifted slightly downward and flattened the NMDA concentration–effect curve if the cortical slices were pretreated with NMDA in the presence or absence of magnesium followed by removal of the NMDA and subsequent stimulation with carbachol. In contrast, cortical slices that had been prepared and treated similarly to the slices used in the PI experiments were very sensitive to the inhibitory effects of magnesium when using the NMDA stimulation of [3H]norepinephrine release assay in the presence or absence of carbachol. Thus, the modulation of PI hydrolysis by NMDA is much less sensitive to inhibition by magnesium than is the stimulation of neurotransmitter release. It is possible that NMDA receptors that mediate the inhibition of PI hydrolysis are coupled to ion channels that do not possess the characteristic voltage‐dependent blockade by Mg2+.
Characterization of Serotonin N‐Acetyltransferase in the Lateral Eye of the Green Frog Rana perezi: Protective Action of EGTA
Authors: Alonso‐Gómez A. L. , Gancedo B. , Alonso‐Bedate M. , Agapito M. T. , Delgado M. J.
Published: October 2006

Abstract: The kinetics of seRotonin N‐acetyltransferase (NAT) from the lateral eye of Rana perezi have been characterized. NAT from ocular tissue reached maximal activity at a phosphate buffer concentration of 250 mM and a pH of 6.5. Reaction linearity was highly conserved within the homogenate fraction range tested (0.033‐0.33). The time course of ocular NAT reaction showed a high linearity at 25 and 35°C. Km and Vmax estimations for acetyl‐CoA at a 10 mM tryptamine concentration were 63.3 μM and 4.42 nmol/h per eye, respectively. Regardless of the acceptor amine (tryptamine or serotonin), the Km was not affected by the acetyl‐CoA concentration (50 or 250 μM), whereas the Vmax was significantly increased at a 250 μM acetyl‐CoA concentration. Ocular NAT showed a higher affinity for serotonin (Km= 20.7 μM) than for tryptamine (Km= 48‐60 μM); Vmax however, was similar for both substrates. Acetyl‐CoA does not protect ocular NAT; in contrast, the use of EGTA (4 mM) in the assay is essential to protect the enzyme because NAT in ocular crude homogenate shows rapid inactivation. This result suggests that intracellular calcium levels are involved in the NAT inactivation mechanisms in frog ocular tissue.
Characterization of Proenkephalin‐Cleaving Proteinases in Bovine Adrenal Chromaffin Granules Using [35S]Proenkephalin Copolymerized into Sodium Dodecyl Sulfate‐Polyacrylamide Gel Electrophoresis
Authors: Roberts Steven F. , Irvine Joseph W. , Lindberg Iris
Published: October 2006

Abstract: Proteinases capable of cleaving proenkephalin into smaller peptides have been identified in bovine adrenal chromaffin granules using [35S]methionine‐labeled recombinant rat proenkephalin as a selective substrate in sodium dodecyl sulfate‐polyacrylamide gel electrophoresis proteinase radiozymography. This technique was used for the screening of subcellular fractions, general characterization of pH optima, and the mechanistic characterization of proteinases with both reversible and irreversible inhibitors. Two enzymes with approximate molecular masses of 76 and 30 kDa were shown to be localized to the highest‐density fractions of chromaffin granules by sucrose density gradient fractionation. Both were enriched in a 1 M NaCl wash of purified chromaffin granule membranes, were active at high pH, and were characterized as serine proteinases based on inhibition by soybean trypsin inhibitor. The 30‐kDa enzyme was also inhibited by diisopropyl fluorophosphate. d‐Phe‐Pro‐Arg‐CH2Cl, and d‐Val‐Phe‐Lys‐CH2Cl and appeared to be the previously described adrenal trypsin‐like enzyme. A third enzyme, of 66 kDa, was also associated with the 1 M NaCl wash of purified chromaffin granule membranes but was not localized exclusively to chromaffin granules in sucrose gradients. This proteinase was found to be Ca2+ activated and inhibited by EDTA but not diisopropyl fluorophosphate, soybean trypsin inhibitor, p‐chloromercuriphenylsulfonic acid, 1,10‐phenanthroline, or pepstatin.
Alterations in the γ‐Aminobutyric Acid‐Gated Chloride Channel Following Transient Forebrain Ischemia in the Gerbil
Authors: Mileson Beth E. , Ehrmann Martha L. , Schwartz Rochelle D.
Published: October 2006

Abstract: The role of inhibitory neurotransmission in selective neuronal degeneration after transient forebrain ischemia was studied by binding of t‐[35S]butylbicyclophosphorothionate ([35S]TBPS) to the γ‐aminobutyric acid (GABA)‐gated chloride channel and measurement of GABAA receptor function in Mongolian gerbil brain. [35S]TBPS binding to the hippocampus, striatum, and cortex quantified by autoradiography and muscimol‐stimulated 36Cl‐ uptake in synaptoneurosomes of the same regions were examined 1, 4, and 29 days after a 5‐min bilateral carotid occlusion. [35S]TBPS binding was decreased in the pyramidal cell dendritic layers, stratum oriens, and stratum lacunosum‐moleculare of the CA1 hippocampus, 4 and 29 days after occlusion, and in the stratum radiatum 29 days after occlusion. [35S]TBPS binding sites in the lateral striatum decreased 47% 4 days after occlusion. At the same time, there was a corresponding decrease in muscimol‐stimulated 36CI‐ uptake in the striatal synaptoneurosomes. Muscimol‐stimulated 36Cl‐ uptake in the hippocampus decreased slightly 4 days after occlusion and more so after 29 days, although these decreases were not significant. No changes were observed in somatosensory cortex at any time point. These data suggest that a portion of GABAA receptors in areas sensitive to ischemic insult are associated with degenerating neurons, whereas other GABAA receptors are spared.
Glutamate Decarboxylases in Nonneural Cells of Rat Testis and Oviduct: Differential Expression of GAD65 and GAD67
Authors: Tillakaratne Niranjala J. K. , Erlander Mark G. , Collard Michael W. , Greif Karen F. , Tobin Allan J.
Published: October 2006

Abstract: γ‐Aminobutyric acid (GABA) and its synthetic enzyme, glutamate decarboxylase (GAD), are not limited to the nervous system but are also found in nonneural tissues. The mammalian brain contains at least two forms of GAD (GAD67 and GAD65), which differ from each other in size, sequence, immunoreactivity, and their interaction with the cofactor pyridoxal 5′‐phosphate (PLP). We used cDNAs and antibodies specific to GAD65 and GAD67 to study the molecular identity of GADs in peripheral tissues. We detected GAD and GAD mRNAs in rat oviduct and testis. In oviduct, the size of GAD, its response to PLP, its immunoreactivity, and its hybridization to specific RNA and DNA probes all indicate the specific expression of the GAD65 gene. In contrast, rat testis expresses the GAD67 gene. The GAD in these two reproductive tissues is not in neurons but in nonneural cells. The localization of brain GAD and GAD mRNAs in the mucosal epithelial cells of the oviduct and in spermatocytes and spermatids of the testis shows that GAD is not limited to neurons and that GABA may have functions other than neurotransmission.
Blood–Brain Barrier Pericytes Are the Main Source of γ‐Glutamyltranspeptidase Activity in Brain Capillaries
Authors: Risau Werner , Dingler Andrea , Albrecht Ursula , Dehouck Marie‐Pierre , Cecchelli Roméo
Published: October 2006

Abstract: Cerebral endothelial cells form the selective permeability barrier between brain and blood by virtue of their impermeable tight junctions and the presence of specific carrier systems. These specialized properties of brain capillaries are reflected in the presence of proteins that are not found in other capillaries of the body. γ‐Glutamyltranspeptidase (GGT) has been widely used as a marker for brain capillaries and differentiated properties of brain endothelial cells. By using histochemical and biochemical methods we have investigated the expression of GGT in isolated capillaries, cultured brain endothelial cells and pericytes, and cocultures of astrocytes and brain endothelial cells. It was surprising that the majority of GGT activity was associated with pericytes, but not endothelial cells, suggesting that GGT is a specific marker for brain pericytes. The remaining GGT activity that was associated with endothelial cells rapidly disappeared from cultured cells but was reinduced in cocultures with astrocytes. Our results emphasize the need for pure endothelial cells for the investigation of blood‐brain barrier characteristics.
Effects of Dopaminergic Transmission Interruption on the D2 Receptor Isoforms in Various Cerebral Tissues
Authors: Martres M. P. , Sokoloff P. , Giros B. , Schwartz J. C.
Published: October 2006

Abstract: We examined the effects of an interruption of dopamine neurotransmission, by either dopamine receptor blockade or degeneration of dopamine neurons by 6‐hydroxydopamine, on the levels of D2 receptor mRNAs. In addition, we evaluated by the polymerase chain reaction (PCR) the relative abundance of the two D2 receptor isoform mRNAs generated by alternative splicing. Daily injections of 4 mg/kg of haloperidol to rats elicited in striatum a rapid and progressive increase in D2 receptor mRNA levels, which reached 70% after a 15‐day treatment. By contrast, there was no apparent change in D2 receptor mRNA levels in cerebral cortex and pons‐medulla, in spite of an increased density of D2 receptor in the former tissue. Using the PCR with primers flanking the alternative exon, we observed that the relative proportion of the shorter receptor isoform (D2s) mRNA was slightly but significantly enhanced in cerebral cortex (17%) and pons‐medulla (18%) after a 15‐day haloperidol treatment. Unilateral degeneration of dopamine neurons induced by local injection of 6‐hydroxydopamine resulted in a marked decrease in levels of total D2 receptor mRNAs in substantia nigra (—79%) and ventral tegmental (—63%) area, two cell body areas. In the substantia nigra, the longer isoform (D2l) mRNA was significantly more decreased in content than the D2s isoform mRNA, so that there was a large enhancement in the relative abundance of the latter (81%). In contrast, the lesion did not result in any significant change in levels of total D2 receptor mRNAs in striatum, but the relative proportion of D2s receptor mRNA tended to decrease—although nonsignificantly—as a result of a tendency of the D2l receptor mRNA abundance to rise. The present study establishes that two distinct processes of D2 receptor gene expression accompany and may contribute to the hypersensitivity known to develop at D2 receptors following either their chronic blockade by neuroleptics or dopamine denervation.
Transcription‐Dependent and ‐Independent Induction of Cerebral Ornithine Decarboxylase
Authors: Zawia N. H. , Bondy S. C.
Published: October 2006

Abstract: Ornithine decarboxylase (ODC: EC 4.1.1.17) is a highly inducible, rate‐limiting enzyme of the polyamine pathway. We have studied the mechanisms that lead to the induction of ODC activity in response to electrical stimulation in three brain regions. Hippocampal ODC activity was found to exhibit much larger elevations than that of the neocortex and the cerebellum. The levels of ODC gene expression were also followed to examine its relationship to the existing regional differences in ODC activity. In the neocortex, there was an elevation of both the ODC mRNA and enzyme activity. However, the hippocampal ODC mRNA level was not increased by electroconvulsive shock. Furthermore, the effects of hormonal changes and seizures on these regional differences in ODC induction were also examined. Adrenalectomy did not affect ODC activity, but pretreatment with the anticonvulsant MK‐801 caused a depression of the induced levels of enzyme activity. Our data suggest that ODC activity in all the brain regions studied is directly elevated by electrically stimulated seizures. However, this induced ODC activity may or may not involve enhanced gene expression.
Nicotine Enhances 1‐Methyl‐4‐Phenyl‐1,2,3,6‐Tetrahydropyridine Neurotoxicity
Authors: Behmand Ramin A. , Harik Sami I.
Published: October 2006

Abstract: Epidemiologic evidence of an inverse relationship between cigarette smoking and Parkinson's disease suggests that a component of cigarette smoke protects against nigrostriatal degeneration. Nicotine, a major component of cigarette smoke, is similar in chemical structure to 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) and is metabolized in part by the same enzymes that detoxify MPTP. We investigated the effect of chronic nicotine on MPTP neurotoxicity in two strains of mice and found that nicotine increases rather than decreases MPTP toxicity. These results are not compatible with the hypothesis that nicotine is that component of cigarette smoke that protects against nigrostriatal degeneration, at least in the MPTP experimental model of Parkinson's disease.
Secretogranin I/Chromogranin B Is a Heparin‐Binding Adhesive Protein
Authors: Chen Ming , Tempst Paul , Yankner Bruce A.
Published: October 2006

Abstract: The major heparin‐binding protein secreted by PC12 cells was purified from conditioned medium. Amino‐terminal sequencing of the purified protein identified it as secretogranin I/chromogranin B (SgI/ChmB). The protein showed the same electrophoretic mobility and biochemical characteristics as previously reported for SgI/ChmB and could be purified in high yield using a simple procedure. In vitro experiments demonstrated that SgI/ChmB effectively promoted cell‐substratum adhesion of NIH 3T3 and PC12 cells and supported neurite outgrowth in primary hippocampal neurons. Thus, SgI/ChmB may be a new member of the family of heparin‐binding extracellular matrix proteins that mediate cell adhesion and support neurite outgrowth.
On the Use of Multiple Probe Insertions at the Same Site for Repeated Intracerebral Microdialysis Experiments in the Nigrostriatal Dopamine System of Rats
Authors: Camp Dianne M. , Robinson Terry E.
Published: October 2006

Abstract: The effects of implantation of a dialysis probe into the striatum of awake rats on indices of dopamine (DA) and serotonin neurotransmission were assessed, first over 24 h following initial insertion of a probe, and then again following reinsertion of a probe at the same site 1 week later. It was found that the basal concentration of DA in dialysate stabilized within 20–40 min after probe implantation, although DA showed a modest decline 24 h later. There was, however, no significant difference in basal DA between two test sessions separated by 1 week. On the other hand, the basal concentrations of the DA metabolites, 3,4‐dihydroxyphenylacetic acid and homovanillic acid, progressively increased for 2–3 h after probe implantation and decreased markedly by 24 h later. Furthermore, in contrast to DA, the DA metabolites decreased even further after the second probe insertion. Amphetamine‐stimulated DA release was also greatly attenuated following the second probe insertion, relative to the first probe insertion. Two probe insertions had only modest effects on the concentration of 5‐hydroxyindoleacetic acid in dialysate, relative to the DA metabolites. It is suggested the effects of two probe insertions on DA metabolism and amphetamine‐stimulated DA release described here are indicative of probe‐induced damage to the nigrostriatal DA system. If this is the case, multiple probe insertions may not provide a feasible strategy for within‐subjects design dialysis experiments over extended periods of time, at least in the DA system of small animals. It is suggested further that a stable basal concentration of DA in dialysate may be an especially poor indicator of the integrity of the dopaminergic input to the striatum.
Brain Mitochondrial DNA Is Not Damaged by Prolonged Cardiac Arrest or Reperfusion
Authors: White Blaine C. , Tribhuwan Rajanikant C. , Van der Laan Douglas J. , DeGracia Donald J. , Krause Gary S. , Grossman Lawrence I.
Published: October 2006

Abstract: Postischemic reperfusion is known to cause iron‐mediated peroxidation of polyunsaturated fatty acids in membranes, including mitochondrial membranes, in the brain cortex. Consequently, we tested the hypothesis that this radical‐mediated damage would extend to DNA. Mitochondrial DNA (mtDNA) was chosen because of its presence at a known site of free radical formation, its sensitivity and ease of assay, and its known lack of any repair systems. In model experiments we utilized endonuclease III or piperidine to amplify topological form conversions in mtDNA damaged by in vitro reactions with hydroxyl radical. We then applied the amplified detection assays to dog brain mtDNA isolated after 2 or 8 h of reperfusion following a 20‐min cardiac arrest. We found that ischemia and reperfusion caused no topological form conversions in mtDNA. Similarly, nucleotide incorporation by a gap‐filling reaction showed no sensitivity to digestion of the mtDNA by exonuclease III, an enzyme known to remove blocked 3’ termini at the site of radical‐generated nicks. Furthermore, the recovery of mtDNA was similar in all experimental groups, suggesting that putatively damaged forms had not been removed by rapid degradation. Thus, despite mitochondrial membrane damage, brain mtDNA does not accumulate oxygen radical damage during postischemic brain reperfusion.
Properties of Muscarinic‐Stimulated Adenylate Cyclase Activity in Rat Olfactory Bulb
Authors: Olianas Maria C. , Onali Pierluigi
Published: October 2006

Abstract: The muscarinic stimulation of adenylate cyclase activity in rat olfactory bulb was characterized, with the aim of elucidating the nature of the molecular mechanism involved. Carbachol (CCh) stimulated the enzyme activity in either crude or purified cell membrane preparations and increased cyclic AMP accumulation in miniprisms of olfactory bulb. The CCh stimulation of adenylate cyclase activity displayed a fast onset and was rapidly reversed by addition of atropine. The stimulation was associated with an increase in the apparent Vmax of the enzyme, with no change in the Km for Mg‐ATP. The affinity of the enzyme for Mg2+ was enhanced by CCh. The muscarinic effect required GTP at concentrations higher than those needed for enzyme stimulation with either l‐isoproterenol or vasoactive intestinal peptide. Moreover, contrary to the β‐adrenergic stimulation, the muscarinic effect disappeared when guanosine 5′‐O‐(3′‐thiotriphosphate) was substituted for GTP. In vivo treatment of olfactory bulbs with pertussis toxin completely prevented the muscarinic stimulation of adenylate cyclase, whereas cholera toxin was without effect. These results indicate that in rat olfactory bulb muscarinic receptors increase adenylate cyclase activity by interacting with a pertussis toxin‐sensitive GTP‐binding protein different from the stimulatory GTP‐binding protein.
Dopaminergic Modulation of Glutamate Release in Striatum as Measured by Microdialysis
Authors: Yamamoto Bryan K. , Davy Sheri
Published: October 2006

Abstract: Glutamate and aspartate are the primary neurotransmitters of projections from motor and premotor cortices to the striatum. Release of glutamate may be modulated by dopamine receptors located on corticostriatal terminals. The present study used microdialysis to investigate the dopaminergic modulation of in vivo striatal glutamate and aspartate release in the striatum of awake‐behaving rats. Local perfusion with a depolarizing concentration of K+ through a dialysis probe into the rat striatum produced a significant increase in the release of glutamate, aspartate, and taurine. The D2 agonist LY171555 blocked the K+‐induced release of glutamate and aspartate, but not taurine, in a concentration‐dependent manner. The D1 agonist SKF 38393 did not alter K+‐induced release of glutamate and taurine, but did significantly decrease aspartate release. Neither agonist had any effect on basal amino acid release. The D2 antagonist (‐)‐sulpiride reversed the inhibitory effects of LY 171555 on K+‐induced glutamate release. These results provide in vivo evidence for a functional interaction between dopamine, the D2 receptor, and striatal glutamate release.
Ischemia‐Induced Translocation of Ca2+/Calmodulin‐Dependent Protein Kinase II: Potential Role in Neuronal Damage
Authors: Aronowski Jaroslaw , Grotta James C. , Waxham M. Neal
Published: October 2006

Abstract: The activities of Ca2+/calmodulin (CaM)‐dependent, Ca2+/phospholipid‐dependent, and cyclic AMP‐dependent protein kinases (CaM‐KII, PKC, and PKA, respectively) were determined in rat brains after global ischemia. Both CaM‐KII and PKC activities were significantly depressed in both hippocampal and cerebral cortical regions of ischemic animals, whereas no change was detected in PKA activity. The loss of CaM‐KII activity was more dramatic and more sustained than the loss of PKC activity and correlated with the duration of ischemia. These decreases in enzyme activity were found in both supernatant and pellet fractions from crude homogenates. When the supernatant and pellet were analyzed for the amount of CaM‐KII 50‐kDa protein, a significant decrease was detected in supernatant fractions that paralleled a gain in the amount of CaM‐KII in the pellet. Thus, the loss of CaM‐KII activity in the supernatant can be explained by translocation of the enzyme to the pellet. Whether inactivation of CaM‐KII occurs during or after the enzyme translocates from the supernatant to the pellet is unknown. Our results indicate that loss in CaM‐KII activity parallels neuronal damage associated with ischemia; down‐regulation of CaM‐KII activity coincided with translocation of the enzyme to the particulate fraction, and it is proposed that this may be, in fact, a mechanism for controlling excessive CaM‐KII phosphorylation.
Superoxide Dismutase, Catalase, and Glutathione Peroxidase Activities in Copper/Zinc‐Superoxide Dismutase Transgenic Mice
Authors: Przedborski S. , Jackson‐Lewis V. , Kostic V. , Carlson E. , Epstein C. J. , Cadet J. L.
Published: October 2006

Abstract: Copper/zinc‐superoxide dismutase (CuZn‐SOD) transgenic mice overexpress the gene for human CuZn‐SOD. To assess the effects of the overexpression of CuZn‐SOD on the brain scavenging systems, we have measured the activities of manganese‐SOD (Mn‐SOD), catalase, and glutathione peroxidase (GSH‐Px) in various regions of the mouse brain. In nontransgenic mice, cytosolic CuZn‐SOD activity was highest in the caudate–putamen complex; this was followed by the brainstem and the hippocampus. The lowest activity was observed in the cerebellum. In transgenic mice, there were significant increases of cytosolic CuZn‐SOD activity in all of these regions, with ratios varying from a twofold increase in the brainstem to 3.42‐fold in the cerebellum in comparison with nontransgenic mice. Particulate Mn‐SOD was similarly distributed in all brain regions, and its levels also were significantly increased in superoxide dismutase (SOD)‐transgenic mice. In the brains of nontransgenic mice, cytosolic catalase activity was similar in all brain regions except the cortex, which showed <50% of the activity observed in the other regions. In transgenic mice, cytosolic catalase activity was significantly increased, with the cortex showing the greatest changes (133%) in comparison with nontransgenic mice. The smallest increases were observed in the hippocampus (34%). In contrast to what was observed for SOD and catalase, there were no significant changes in cytosolic GSH‐Px activity in any of the brain regions examined. The present results indicate that, in addition to displaying marked increases in the levels of brain CuZn‐SOD activity, SOD‐transgenic mice also exhibit increases in other enzymes that scavenge oxygen‐based radicals. We also found that aminotriazole caused significantly greater inhibition of brain catalase activity in SOD‐transgenic mice (67.6%) than in nontransgenic littermates (43.6%). The latter finding provides evidence for a higher production of H2O2 in the brains of SOD‐transgenic mice. When taken together, these results further support the use of these animals to assess the role of free radicals in ischemia/reperfusion and in the aging process.
On the Stability of Messenger RNA and Ribosomal RNA in the Brains of Control Human Subjects and Patients with Alzheimer's Disease
Authors: Ross B. M. , Knowler J. T. , McCulloch J.
Published: October 2006

Abstract: The levels of the mRNAs encoding the G protein subunits Gsα, Gβ1, and Gβ2 were measured by northern blotting in the frontal cortex and hippocampus of control subjects and of patients with a clinical and histopathological diagnosis of dementia of the Alzheimer type (DAT). There was no significant difference, in either brain region, between the control and DAT groups for any of the G protein mRNAs measured. The degree of intersubject variability was very high, e.g., Gsα mRNA in the frontal cortex (mean optical density ± SD) was 405 ± 342 in the control group versus 305 ± 207 in the DAT group. The extent of generalised RNA degradation was assessed by detecting the breakdown products of 28S rRNA. RNA degradation was present in tissue samples from every human subject studied. The extent of 28S rRNA degradation in each subject was found to be related to the levels of G protein mRNA detected. The degree of RNA degradation in human subjects was found to be very variable and unaffected by the presence of DAT. RNA degradation correlated poorly with postmortem interval and this was confirmed by a controlled study of postmortem degradation in rat tissue. The possibility that the relative hypoxia and ischaemia in patients immediately before death could influence RNA degradation is discussed. The variable extent of RNA degradation means that great care must be taken to ensure the validity of RNA analyses undertaken in human postmortem brain, particularly when techniques are employed (such as in situ hybridisation) that themselves give no indication of RNA integrity.
AT1 Angiotensin Receptors Mobilize Intracellular Calcium in a Subclone of NG108–15 Neuroblastoma Cells
Authors: Ransom J. T. , Sharif N. A. , Dunne J. F. , Momiyama M. , Melching G.
Published: October 2006

Abstract: The effects of angiotensin II (AII) and related peptides on the mobilization of internal Ca2+ were studied in a subclone of NG108–15 cells. The subclone, C1, was prepared by fluorescence‐activated cell cloning using a rapid response kinetics and a large response magnitude following stimulation by AII as the selection criteria. Angiotensin I, AII, and angiotensin III (AIII) stimulated Ca2+ mobilization in the C1 cells in a concentration‐dependent manner (1 nM‐100 μM), yielding EC50 values of 437 ± 80 nM (n = 4; slope = 1.6 ± 0.3), 57 ± 8 nM (n = 12; slope = 1.5 ± 0.3), and 36 ± 5 nM (n = 7; slope = 1.4 ± 0.3), respectively. AIII was significantly more potent than AII (p < 0.05). In contrast, Des‐Phe8‐AII, AII‐hexapeptide (AII 3–8), and p‐NH2‐Phe6‐AII (1–10 μM) were inactive as agonists. Although the effects of AII and AIII in C1 and parent NG108–15 cells were totally inhibited by the AT1 receptor‐selective nonpeptide antagonist, DUP‐753 (0.3–1 μM), the AT2‐selective antagonists, EXP‐655 and CGP42112A (1–10 μM), failed to block the effects of AII. DUP‐753 (0.3–100 nM) produced dextral shifts of the AII‐induced concentration‐response curves and yielded an estimated affinity constant (pA2) of 8.5 ± 0.2 (n = 16) using single‐point analysis involving different concentrations of DUP‐753. These data compared well with those obtained for the inhibition of AII‐induced aortic contractions by DUP‐753 (pA2= 8.5) reported previously by others. These data strongly support the identification of an AT1‐angiotensin receptor subtype in the NG108–15. C1 subclone that is coupled to a Ca2+ mobilization signal transduction mechanism.
Decreased Density of Presynaptic α2‐Adrenoceptors in Postmortem Brains of Patients with Alzheimer's Disease
Authors: Javier Meana J. , Barturen Fernando , Asier Garro M. , García‐Sevilla Jesós A. , Fontán Aurora , Zarranz Juán J.
Published: October 2006

Abstract: The full agonist [3H]bromoxidine (UK 14304) was used to quantitate α2‐adrenoceptors in postmortem brains of patients with Alzheimer's disease. The effects of aging and human serum Cohn fraction IV on [3H]bromoxidine binding were also assessed. In patients with Alzheimer's disease, the binding capacity (Bmax) of [3H]bromoxidine was lower in the frontal cortex (37%), hypothalamus (33%), and cerebellum (52%) than in matched controls. In the hippocampus, amygdala, and head of caudate, the binding capacities (Bmax) were unchanged. Quantitative autoradiographic analyses with [3H]bromoxidine confirmed the existence of a marked reduction (55–60%) in α2A‐adrenoceptor density in the frontal cortex (layers I and III). In patients with dementia who did not meet neuropathological criteria for Alzheimer's disease, the density of α2‐adrenoceptors was unchanged. In control subjects, the density of α2A‐adrenoceptors in the frontal cortex showed a significant negative correlation with age at death. The inhibitory effect of human serum Cohn fraction IV on [3H]bromoxidine was very similar in control subjects and patients with Alzheimer's disease. The observed decrease in the density of brain α2‐adrenoceptors in Alzheimer's disease may represent direct biochemical evidence of a presynaptic location of this receptor on noradrenergic nerve terminals in the human CNS.
Astrocyte Heterogeneity: Endogenous Amino Acid Levels and Release Evoked by Non‐N‐Methyl‐D‐Aspartate Receptor Agonists and by Potassium‐Induced Swelling in Type‐1 and Type‐2 Astrocytes
Authors: Levi Giulio , Patrizio Mario
Published: October 2006

Abstract: The aim of the present study was to determine whether endogenous amino acids are released from type‐1 and type‐2 astrocytes following non‐N‐methyl‐D‐aspartate (NMDA) receptor activation and whether such release is related to cell swelling. Amino acid levels and release were measured by HPLC in secondary cultures from neonatal rat cortex, highly enriched in type‐1 or type‐2 astrocytes. The following observations were made. (a) The endogenous level of several amino acids (glutamate, alanine, glutamine, asparagine, taurine, serine, and threonine) was substantially higher in type‐1 than in type‐2 astrocytes. (b) The spontaneous release of glutamine and taurine was higher in type‐1 than in type‐2 astrocytes; that of other amino acids was similar. (c) Exposure of type‐2 astrocyte cultures to 50 μM kainate or quisqualate doubled the release of glutamate and caused a lower, but significant increase in that of aspartate, glycine, taurine, alanine, serine (only in the case of kainate), and glutamine (only in the case of quisqualate). These effects were reversed by the antagonist CNQX. (d) Exposure of type‐1 astrocyte cultures to 50–200 μM kainate or 50 μM quisqualate did not affect endogenous amino acid release, even after treating the cultures with dibutyryl cyclic AMP. (e) Exposure of type‐1 or type‐2 astrocyte cultures to 50 mM KCl (replacing an equimolar concentration of NaCl) enhanced the release of taurine > glutamate > aspartate. The effect was somewhat more pronounced in type‐2 than in type‐1 astrocytes. Veratridine (50 μM) did not cause any increase in amino acid release. (f) The release of amino acids induced by high [K+] appeared to be related to cell swelling, in both type‐1 and type‐2 astrocytes. Swelling and K+‐induced release were somewhat higher in type‐2 than in type‐1 astrocytes. In contrast, neither kainate nor quisqualate caused any appreciable increase in cell volume. It is concluded that non‐NMDA receptor agonists stimulate the release of several endogenous amino acids (some of which are neuroactive) from type‐2 but not from type‐1 astrocytes. The effect does not seem to be related to cell swelling, which causes a different release profile in both type‐1 and type‐2 astrocytes. The absence of kainate‐ and quisqualate‐evoked release in type‐1 astrocytes suggests that the density of non‐NMDA receptors in this cell type is very low.
Angiotensin‐Induced Cyclic GMP Production Is Mediated by Multiple Receptor Subtypes and Nitric Oxide in N1E‐115 Neuroblastoma Cells
Authors: Zarahn E. D. , Ye X. , Ades A. M. , Reagan L. P. , Fluharty S. J.
Published: October 2006

Abstract: Angiotensin II (AngII) elicited a rapid and dose‐related production of intracellular cyclic GMP (cGMP) in murine neuroblastoma N1E‐115 cells. The agonist‐induced rise in cGMP levels was blocked in a monophasic fashion by the AT1‐selective antagonist DuP 753 or the nonselective antagonist [Sarc1,Ile8]‐AngII, and both antagonists produced complete inhibition of the cGMP response elicited by submaximal concentrations of AngII. In contrast, the AT2‐selective antagonist CGP 42112A inhibited the cGMP response biphasically. At lower antagonist concentrations, agonist‐induced cGMP production was only partially inhibited, whereas complete inhibition was observed only when the concentration of CGP 42112A was increased sufficiently to interact with both AT1 and AT2 receptor subtypes. AngII‐also increased inositol trisphosphate (InsP3) levels in N1E‐115 cells. However, the InsP3 response was mediated exclusively by the AT1 receptor subtype because it was inhibited by lower, AT1 selective concentrations of DuP 753, whereas only higher, nonselective concentrations of CGP 42112A were effective. Finally, the stimulatory effects of AngII on cGMP production appeared to be mediated by the intracellular formation of nitric oxide in that they were attenuated by the nitric oxide synthase inhibitor, N‐monomethyl‐L‐arginine. Collectively, these results suggest that the AngII‐elicited rise in cGMP levels may require an interaction between AT1‐mediated mobilization of intracellular Ca2+, as well as some partial role of AT2 receptors.
1‐Aminocyclopentane‐trans‐1,3‐Dicarboxylic Acid Induces Glutamine Synthetase Activity in Cultured Astrocytes
Authors: Miller Stephan , Cotman Carl W. , Bridges Richard J.
Published: October 2006

Abstract: In this study we have investigated the effect of excitatory amino acids on the activity of glutamine synthetase, a glial‐specific enzyme that plays a key role in the regulation of glutamate concentration in the CNS. We found that of L‐glutamate, N‐methyl‐D‐aspartate, α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionate, kainate, and 1‐aminocyclopentane‐trans‐1,3‐dicarboxylic acid (trans‐ACPD), only the metabotropic glutamate receptor agonist trans‐ACPD had an effect on glutamine synthetase specific activity in cultures of rat type I cortical astrocytes. Exposure of astrocytes to 1.0 mMtrans‐ACPD for 24 h resulted in an increase in glutamine synthetase activity to 149 ± 11% of that in control cultures. This effect was concentration dependent, stereoselective, and blocked by cycloheximide. In addition, the increase in glutamine synthetase activity occurred at lower concentrations of trans‐ACPD that did not produce morphological alterations or lysis of the astrocytes as measured by the lactate dehydrogenase content. These findings are consistent with the hypothesis that activation of the metabotropic excitatory amino acid receptor in astrocytes is coupled to the regulation of an enzyme essential to the metabolism and recycling of the excitatory transmitter L‐glutamate.
Sensitization of Dopamine‐Stimulated Adenylyl Cyclase in the Striatum of 1‐Methyl‐4‐Phenyl‐1,2,3,6‐Tetrahydropyridine‐Treated Rhesus Monkeys and Patients with Idiopathic Parkinson's Disease
Authors: Pifl Christian , Nanoff Christian , Schingnitz Günther , Schütz Wolfgang , Hornykiewicz Oleh
Published: October 2006

Abstract: Dopamine‐stimulated adenylyl cyclase activity was measured in striatal homogenates of l‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP)‐treated rhesus monkeys and humans with idiopathic Parkinson's disease and compared with the activity in control tissue. No differences between parkinsonian and control tissue were found in the presence of 20 mM NaCl. However, when 120 mM NaCl was included in the assay medium, a significantly higher increase in the Vmax of dopamine‐stimulated adenylyl cyclase activity was observed in the caudate of MPTP‐parkinsonian rhesus monkeys and the putamen of patients with idiopathic Parkinson's disease. No such sensitization was seen in the MPTP‐treated rhesus putamen or human Parkinson's disease caudate tissue. A role of D2 receptors in this sensitization could be ruled out by the concomitant use of the D2 antagonist l‐sulpiride and by [3H]spiperone saturation analysis of the D2 receptor density, which was found at control level in the caudate tissue of MPTP‐treated rhesus monkeys. Similarly, on the basis of saturation binding with the D1 selective ligand 125I‐SCH 23982, there was no difference in caudate nucleus D1 receptor densities between control and MPTP‐treated monkeys. Our results point to a region‐specific functional sensitization of D1 receptors as a consequence of severe dopaminergic denervation of the striatum and suggest the possibility of a therapeutic potential of a D1 agonist with full intrinsic activity in Parkinson's disease.
Molecular Characterisation and Structural Relationship of the Synapse‐Enriched Glycoproteins gp65 and gp55
Authors: Willmott Tim , Skitsa Ioulia , Hill Irene , Mummery Rosemary , Beesley Philip W.
Published: October 2006

Abstract: gp65 and gp55 are glycoprotein components of CNS synapses that are recognised by a single monoclonal antibody, SMgp65. This antibody has now been used to investigate the molecular properties of these two glycoproteins and the structural relationship between them. Both gp65 and gp55 occur in most brain regions as doublets of apparent molecular masses of 63 and 67 kDa, and 52 and 57 kDa, respectively. Striatal samples, however, are enriched in a novel gp65 iso‐form of 69 kDa. Removal of oligosaccharide residues from gp65 and gp55 with trifluoromethanesulphonic acid shows that gp65 and gp55 are composed of single polypeptide chains of 40 and 28 kDa, respectively. Removal of sialic acid residues with neuraminidase lowers the apparent molecular mass of both glycoproteins by 5–6 kDa. Triton X‐114 phase partitioning and alkaline extraction of synaptic membranes indicate that both gp65 and gp55 are integral membrane glycoproteins. Treatment of synaptic membranes with phosphatidylinositol‐specific phospholipase C does not solubilise either glycoprotein. One‐dimensional peptide and epitope maps obtained by digestion of gp65 and gp55 with endoproteinase lys C or subtilisin are consistent with a close structural relationship between the two molecules. Tryptic digestion of samples enriched in gp65 and/or gp55 results in the formation of a novel immunoreactive 53‐kDa species that is resistant to further trypsin degradation except in the presence of 0.1% (wt/vol) sodium dodecyl sulphate. Trypsin treatment of cultures of forebrain neurones in situ lowers the apparent molecular mass of gp65 to 53 kDa. These results confirm the structural similarity ofgp6 5 andgp5 5 and suggest that the major difference between the two glycoproteins is likely to be a single 12‐kDa cell surface‐located polypeptide fragment.
Sequences That Direct Rat Tyrosine Hydroxylase Gene Expression
Authors: Fung Brenda P. , Ok Yoon Sung , Chikaraishi Dona M.
Published: October 2006

Abstract: Investigation of neuroendocrine genes has revealed that transcription is regulated via multiple DN A binding sites, including the cyclic AMP response element (CRE). We show here that for the neuronal and chromaffin‐specific gene tyrosine hydroxylase (TH), a 70‐bp region (‐229 to ‐160) lacking the CRE is sufficient, in either orientation, to confer levels of chloramphenicol acetyltransferase reporter expression equivalent to or greater than that conferred by 4.8 kb of the rat TH enhancer/promoter region. The 70‐bp region contains potential binding sites for AP2, API, E2A/MyoD, and POU transcription factors, and functions when linked to the TH promoter, but not when joined to a heterologous RSV promoter. This demonstrates that promoter as well as enhancer elements are important for TH expression. In gel‐shift assays, the 70‐bp fragment forms a cell type‐specific complex with nuclear extracts from TH‐expressing cells, which is effectively competed by an oligonucleotide containing AP2, AP1, and E2A/MyoD (E box) sites, but not by one containing the POU site. These data suggest that the AP2, API, and/or E box sites may be involved in forming the cell‐specific complex. Although it lacks an authentic CRE, the 70‐bp region also mediated a twofold transcriptional response to forskolin, equivalent to that found with the endogenous gene. A different region (‐60 to ‐29) bearing a consensus CRE mediated a sixfold increase in transcription in response to forskolin, but only minimally activated basal transcription from the TH promoter in the absence of forskolin.
A Molecular Mechanism for the Induction of Neurofilament Bundling by Aluminum Ions
Authors: Leterrier J. F. , Langui D. , Probst A. , Ulrich J.
Published: October 2006

Abstract: Al induces neurofibrillary tangles in the perikaryon of neurons in vivo and in culture. The effect of Al ions complexed with maltol, a plant‐derived ligand of Al, on purified neurofilament preparations was studied in vitro. The binding of Al to the arm‐like projections of the high (H)‐ and medium (M)‐molecular‐weight neurofilament subunits causes a conformational change of the molecule (intrafilamentous reaction), characterized by an altered migration on sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE). In addition, Al compounds strongly stimulate the interaction between neurofilaments (interfilamentous reaction). The possibility that phosphate groups of the H and M sidearms are involved in binding Al ions is discussed with regard to the migration on SDS‐PAGE of dephosphorylated neurofilaments incubated with Al compounds and the alteration by Al ions of neurofilament phosphorylation in vitro by the associated kinase. Immunoblotting analysis of neurofilaments in cultivated neurons intoxicated with Al compounds revealed a similar Al‐dependent alteration of the neurofilament subunit conformation. This result suggests that the mechanism of Al‐induced bundling of neurofilaments derived from in vitro studies might be involved in the formation of tangles in situ.
Purification and Characterization of Naturally Occurring Benzodiazepine Receptor Ligands in Rat and Human Brain
Authors: Rothstein Jeffrey D. , Garland William , Puia Gulia , Guidotti Alessandro , Weber Richard J. , Costa Erminio
Published: October 2006

Abstract: Chemicals that are active at the benzodiazepine receptor (endozepines) are naturally present in the CNS. These substances are present in tissue from humans and animals and in plants and fungi. Using selective extraction protocols, HPLC purification, receptor binding displacement studies, and selective anti‐benzodiazepine antibodies, we have identified six or seven peaks of endozepines in rat and human brain. All material could competitively displace [3H]flunitrazepam binding to cerebellar benzodiazepine binding sites. Two peaks also competitively displaced Ro 5‐4864 binding to the mitochondrial benzodiazepine binding site. Total amounts of brain endozepines were estimated to be present in potentially physiological concentrations, based on their ability to displace [3H]flunitrazepam binding. Although endozepine peaks 1 and 2 had HPLC retention profiles similar to those of nordiazepam and diazepam, respectively, gas chromatography‐mass spectrometry as well as high‐performance TLC revealed biologically insignificant amounts of diazepam (< 0.02 pg/g) and nordiazepam (<0.02 pg/g) in the purified material. Electrophysiologically, some purified endozepines positively modulated γ‐aminobutyric acid (GABA) action on Cl− conductance, monitored in patch‐clamped cultured cortical neurons or in mammalian cells transfected with cDNA encoding various GABAA receptor subunits. These studies demonstrate that mammalian brains contain endozepines that could serve as potent endogenous positive allosteric modulators of GABAA receptors.
Stimulus‐Induced Accumulation of Inositol Tetrakis‐, Pentakis‐, and Hexakisphosphates in N1E‐115 Neuroblastoma Cells
Authors: Sasakawa Nobuyuki , Nakaki Toshio , Kashima Reiko , Kanba Shigenobu , Kato Ryuichi
Published: October 2006

Abstract: When [3H]inositol‐prelabelled N1E‐115 cells were stimulated with carbamylcholine (CCh) (100 μM), high K+ (60 mM), and prostaglandin E, (PGE,) (10 μM), a transient increase in [3H]inositol pentakisphosphate (InsP5) accumulation was observed. The accumulation reached its maximum level at 15 s and had declined to the basal level at 2 min. CCh, high K+, and PGE, also caused accumulations of [3H]inositol 1,4,5‐trisphosphate [Ins(1,4,5)P3], [3H]inositol 1,3,4,6‐tetrakisphosphate [Ins(1,3,4,6)P4], and 13H]inositol hexakisphosphate (InsP6). Muscarine and CCh induced accumulations of [3H]Ins(1,4,5)P3, [3H]‐Ins(1,3,4,6)P4, [3H]InsP5, and [3H]InsP6 with a similar potency and exerted these maximal effects at 100 μM, whereas nicotine failed to do so at 1 mM. With a slower time course, CCh, high K+, and PGE1 caused accumulations of [3H]‐inositol 1,3,4‐trisphosphate [Ins(1,3,4)P3] and [3H]inositol 1,3,4,5‐tetrakisphosphate [Ins(1,3,4,5)P4]. In an N1E‐115 cell homogenate, [3H]Ins(1,4,5)P3, [3H]Ins(1,3,4,5)P4, and [3H]Ins(1,3,4)P3 were converted to [3H]InsP5 through [3H]‐Ins(1,3,4,6)P4. The above results indicate that Ins(1,3,4,6)P4, InsP5, and InsP6 are rapidly formed by several kinds of stimulants in N1E‐115 cells.
Selective Release of Spermine and Spermidine from the Rat Striatum by N‐Methyl‐d‐Aspartate Receptor Activation In Vivo
Authors: Fage D. , Voltz C. , Scatton B. , Carter C.
Published: October 2006

Abstract: The intrastriatal infusion of N‐methyl‐d‐aspartate (NMDA; 250–1,000 μM) via a dialysis cannula in anesthetized rats resulted in a marked and rapid increase in the concentrations of spermine and spermidine recovered in the dialysate. Extracellular concentrations of NMDA‐released spermine and spermidine were calculated to be in the low micromolar range. Putrescine levels were not significantly affected by NMDA. The effects of NMDA (500 μM) were blocked by the previous systemic injection of MK‐801 (3 mg/kg, i.p.) but were insensitive to the intrastriatal infusion of tetrodotoxin (1 μM). Intrastriatally infused kainate or quisqualate (1,000 μM) did not increase polyamine levels in the dialysate. Spermine and spermidine dialysate levels were also significantly increased by the infusion of high concentrations of K+ (>100 mM), although the effects of K+ were considerably less marked than those of NMDA. Striatal polyamines are released into the extracellular space specifically by NMDA receptor activation. Because of their multiple effects on receptor‐ and voltage‐operated cation channels, polyamines that are released by NMDA receptor activation may play an important role in phenomena already attributed to NMDA receptor stimulation, such as long‐term potentiation, synaptic plasticity, and neurotoxicity.