bluuebunny/crossref_metadata_2025_split huggingface dataset:
Acetylation of Serotonin in the Rabbit Pineal Gland: An N‐Acetyltransferase with Properties Distinct from NAT1 and NAT2 Is Responsible
Authors: Heim M. H. , Blum M. , Beer M. , Meyer U. A. 
Published: October 2006

AbstractTwo rabbit arylamine N‐acetyltransferases (NAT1 and NAT2, EC 2.3.1.5) have been cloned and characterized recently in this laboratory. They catalyze the acetylation of primary arylamine and hydrazine drugs and other substrates in the liver, including sulfamethazine, ρ‐aminosalicylic acid, and ρ‐aminobenzoic acid. In the pineal gland, serotonin is metabolized to N‐acetylserotonin by an unknown N‐acetyltransferase. Similarity of the liver enzymes and the pineal gland arylalkylamine N‐acetyltransferase (AA‐NAT) has been suggested, because pineal gland homogenates were shown to metabolize arylamine substrates as ρ‐phenetidine, aniline, or phenylethylamine, and liver homogenates or partially purified liver enzyme preparations catalyzed the N‐acetylation of serotonin. The present study was undertaken to elucidate the possible role of NAT1 or NAT2 in serotonin acetylation in the pineal gland. We transiently expressed rNAT1 and rNAT2 genes in COS cells, studied the kinetics of the enzymes produced with various substrates, and compared these data with activities of rabbit pineal glands and livers. These enzymatic studies were complemented with western blot analysis with antibodies against NAT1 and NAT2. Cross‐hybridization of rNAT1 or rNAT2 to the gene for the pineal gland AA‐NAT was tested by Southern blot studies of genomic rabbit DNA. Our results indicate that although NAT1 is expressed in the pineal gland, it is not involved in the physiologically important step of N‐acetylation of serotonin.
Amphiphilic and Nonamphiphilic Forms of Bovine and Human Dopamine β‐Hydroxylase
Authors: Bon Suzanne , Lamouroux Annie , Vigny Annette , Massoulié Jean , Mallet Jacques , Henry Jean‐Pierre 
Published: October 2006

AbstractWe show that human and bovine dopamine β‐hydroxylases (DBH) exist under three main molecular forms: a soluble nonamphiphilic form and two amphiphilic forms. Sedimentation in sucrose gradients and electrophoresis under nondenaturing conditions, by comparison with acetylcholin‐esterase (AChE), suggest that the three forms are tetramers of the DBH catalytic subunit and bind either no detergent, one detergent micelle, or two detergent micelles. By analogy with the Gna4 and Ga4 AChE forms, we propose to call the nonamphiphilic tetramer Dna4 and the amphiphilic tetramers Da4I and Da4II. In addition to the major tetrameric forms, DBH dimers occur as very minor species, both amphiphilic and nonamphiphilic. Reduction under nondenaturing conditions leads to a partial dissociation of tetramers into dimers, retaining their amphiphilic character. This suggests that the hydrophobic domain is not linked to the subunits through disulfide bonds. The two amphiphilic tetramers are insensitive to phosphatidylinositol phospholipase C, but may be converted into soluble DBH by proteolysis in a stepwise manner; Da4II ‐Da4I‐S. Incubation of soluble DBH with various phospholipids did not produce any amphiphilic form. Several bands corresponding to the catalytic subunits of bovine DBH were observed in sodium dodecyl sulfate‐polyacrylamide gel electrophoresis, but this multiplicity was not simply correlated with the amphiphilic character of the enzyme. In the case of human DBH, we observed two bands of 78 and 84 kDa. As previously reported by others, the presence of the heavy subunit characterizes the amphiphilic forms of the enzyme. We discuss the nature of the hydrophobic domain, which could be an uncleaved signal peptide. and the organization of the different amphiphilic and nonamphiphilic DBH forms. We present two models in which dimers may possess either one hydrophobic domain or two domains belonging to each subunit; in both cases, a single detergent micelle would be bound per dimer.
The Synthesis and Release of Acetylcholine by Depolarized Hippocampal Slices Is Increased by Increased Choline Available In Vitro Prior to Stimulation
Authors: Wecker Lynn 
Published: October 2006

AbstractThe objective of these experiments was to determine whether preincubating hippocampal slices with choline provides precursor that can be used during a subsequent incubation to support or enhance the synthesis of acetylcholine (ACh). Slices were preincubated for 60 min with 0, 10, 25, or 50 μM choline, washed, resuspended, and then incubated for 10 min in choline‐free buffer containing 4.74 (Krebs‐Ringer bicarbonate, KRB) or 25 mM KC1. The tissue contents of ACh and choline were determined prior to and after the preincubation, as well as after the incubation; the amounts of ACh and choline released were measured, and ACh synthesis was calculated. Preincubation in the absence of choline increased the tissue content of ACh to 242% of original levels; preincubation with 10 μM choline did not lead to a further increase, but preincubation with 25 or 50 μM choline increased the ACh content to 272% of original levels, significantly greater than that of slices preincubated with either 0 or 10 μM choline. When tissues were subsequently incubated for 10 min with either KRB or 25 μM KC1, ACh release from slices preincubated with 50 μM choline was greater than from slices preincubated with 0, 10, or 25 μM choline. Incubation of slices with KRB did not alter the tissue content of ACh, but when tissues were incubated with 25 mM KC1, the ACh content of slices preincubated with 0 or 10 μM choline decreased significantly, whereas that of slices preincubated with 25 or 50 μM choline did not. Although ACh synthesis by slices incubated with KRB did not differ significantly among groups, synthesis by slices incubated with 25 mM KC1 was significantly greater for tissues preincubated with 50 μM choline than for those preincubated with 0, 10, or 25 μM choline. Preincubation in the absence of choline decreased choline levels by 43%, whereas preincubation with 50 μM choline increased levels by 55%; choline levels were unaltered by preincubation with 10 μM choline. Following incubation with 25 mM KCl, the choline content of slices preincubated with 50 μM choline decreased by 0.59 nmol, whereas the content of slices preincubated with 10 μM choline decreased by 0.33 nmol, suggesting that the former used more of its tissue choline to support ACh synthesis; choline levels did not change in slices preincubated in the absence of choline. These results suggest that prior exposure of hippocampal slices to choline provides precursor that is used during a subsequent incubation with 25 mM KCl to support the synthesis of ACh.
Ontogeny of the Benzodiazepine Receptor in Human Brain: Fluorographic, Immunochemical, and Reversible Binding Studies
Authors: Reichelt Ralf , Hofmann Dietmar , Födisch Hans‐Jörg , Möhler Hanns , Knapp Michael , Hebebrand Johannes 
Published: October 2006

AbstractThe prenatal and postnatal human ontogeny of the central benzodiazepine receptor was investigated in six different brain regions between week 24 postconception and age 14 years. Binding studies, which were performed with [3H]flunitrazepam ([3H]FNZ), revealed a steep increase in receptor density postnatally in frontal cortex and cerebellum. Bmax values were higher in medulla oblongata, pons, and thalamus than in cortex and cerebellum up to week 26. After that, receptor densities declined significantly in medulla and olive. The same tendency was apparent in pons, whereas receptor density remained unchanged in thalamus. The early ontogeny of the benzodiazepine receptor was also evaluated in fluorographs ([3H]FNZ) and immunoblots using the αrsubunit‐specific monoclonal antibody (mAb) bd‐24. Specific radiolabeled proteins with molecular weights of 53K and 59K were visible in cortical membranes from gestational week 8, the earliest time investigated. During further development, the intensity of the 53K band increased without changes in the 59K band. As in other species, postmortem proteolysis in human brain led to a specifically labeled peptide of 47K. The mAb bd‐24 immunolabeled only the 53K protein and the 47K peptide.
Nondestructive Detection of Glutamate by 1H Nuclear Magnetic Resonance Spectroscopy in Cortical Brain Slices from the Guinea Pig: Evidence for Changes in Detectability During Severe Anoxic Insults
Authors: Kauppinen Risto A. , Williams Stephen R. 
Published: October 2006

Abstract 31P and 1H nuclear magnetic resonance spectroscopy (NMR) was used to study the metabolism of intact superfused cortical brain slices during normoxia and anoxia. Attention was focused on quantification of 1H NMR‐detected glutamate by a water‐suppressed spin‐echo method, using N‐acetyl aspartate as an internal concentration reference. To quantify the 1H NMR signals, the spin‐spin relaxation times and saturation effects were estimated for given metabolites. In addition, absolute concentrations of metabolites were determined by biochemical methods from acid extracts of the preparations after NMR experiments. Under aerobic conditions, 1H NMR detected 79% of the glutamate determined biochemically from the brain slice extracts. During anoxia in the absence of glucose when a severe energetic failure was evident, both 1H NMR and biochemical assays gave closely matching levels for glutamate. We conclude that in the brain cortex 21% of glutamate is located in an intracellular compartment in which this amino acid does not contribute to the 1H NMR signal. However, during severe anoxia an intracellular reorganization occurs increasing the detectability of this amino acid neurotransmitter by NMR.
Complex Interactions Between Polyamines and Calpain‐Mediated Proteolysis in Rat Brain
Authors: Najm Imad , Vanderklish Peter , Etebari Amir , Lynch Gary , Baudry Michel 
Published: October 2006

AbstractPolyamine synthesis is induced by various extracellular signals, and it is widely held that this biochemical response participates in cell growth and differentiation. Certain of the triggers for synthesis in brain tissues also increase the breakdown of high‐molecular‐weight structural proteins, apparently by activating calcium‐dependent proteases (cal‐pains). The present experiments tested the possibility that calpain activity is modulated by polyamines. Spermine, spermidine, and putrescine all increased calcium‐dependent proteolysis of [14C]casein by soluble fractions of rat brain. The order of potency was spermine > spermidine > putrescine, with apparent affinities of 30, 300, and 6.000 μM, respectively. Each of the three polyamines at physiological concentrations also potentiated the calcium‐dependent breakdown of two endogenous high‐molecular‐weight structural proteins known to be substrates of calpain, in both supernatant and membrane fractions. The thiol protease inhibitor leupeptin, a known calpain inhibitor, also inhibited calcium‐dependent proteolysis in the presence and absence of polyamines. The polyamines did not increase the activity of purified calpain I or calpain II determined with either [14C]casein or purified spectrin as the substrate, nor did they interfere with the inhibitory effects of calpastatin, an endogenous inhibitor of calpain. However, polyamines potentiated the stimulation of endogenous but not purified calpain activity produced by an endogenous calpain activator. These results suggest a rote for polyamines in protein degradation as well as protein synthesis.
Postnatal Age and Protein Tyrosine Phosphorylation at Synapses in the Developing Rat Brain
Authors: Cudmore Stephen B. , Gurd James W. 
Published: October 2006

AbstractThe relationship between postnatal age and protein tyrosine kinase activity in synaptosomes prepared from the rat forebrain was studied. Synaptosomal particulate and soluble fractions, as well as total homogenates, the cell soluble fraction, and P3, were prepared from rats ranging in postnatal age from 5 to 60 days and analyzed for (a) tyrosine kinase activity using polyglutamyltyrosine (4:1) as the substrate, (b) the presence of endogenous substrates for tyrosine phosphorylation using polyclonal antibodies specific for phosphotyrosine, and (c) levels of pp60src. Enzyme activity, expressed per milligram of protein, in the total homogenate, P3, and both the cell and synaptosomal soluble fractions was highest in the brains of young animals (postnatal days 5–10) and decreased thereafter to adult levels. In contrast, tyrosine kinase activity in the synaptosomal particulate fraction exhibited a unique biphasic developmental profile, increasing to maxima at postnatal days 10 and 20 before decreasing to adult values. Endogenous substrates for tyrosine phosphorylation were identified by incubating subcellular fractions with 2 mM ATP in the presence of sodium orthovanadate and probing nitrocellulose blots of proteins separated by gel electrophoresis with antiphosphotyrosine antibodies. Several phosphotyrosine‐containing proteins were detected in the synaptosomal particulate and P3 fractions, including proteins of Mr 180K, 145K, 120K, 100K, 77K, 68K, 62K, 54K, 52K, and 42K. In the cell soluble fraction a protein doublet of Mr 54/52K and a 120K protein were the major phosphotyrosine‐containing proteins. The 54/52K doublet was the major protein tyrosine kinase substrate in the synaptosomal soluble fraction. There was a general decrease in the immunostaining of phosphotyrosine‐containing proteins in the P3 fraction after postnatal day 10. In contrast, the tyrosine phosphorylation of several proteins in the synaptosomal particulate fraction increased to maximal levels during the third and fourth week after birth before declining to adult levels. Western blotting using monoclonal antibody 327, specific for pp60src, showed that pp60src levels decreased throughout development in both homogenate and P3 fractions. The concentration of pp60src in the synaptosomal particulate fraction and postsynaptic densities, in contrast, increased 80–100% during the third and fourth weeks before decreasing slowly to adult levels. The results indicate that the expression of tyrosine kinase and the phosphorylation of synaptic proteins on tyrosine residues at the developing synapse are transiently increased during the latter stages of synaptogenesis and suggest that the phosphorylation of proteins on tyrosine residues may be involved in the regulation of synapse formation.
Characterization and Ca2+ Requirement of Histamine‐Induced Catecholamine Secretion in Cultured Bovine Chromaffin Cells
Authors: Goh Yasumasa , Kurosawa Atsushi 
Published: October 2006

AbstractThe stimulation of cultured bovine chromaffin cells with histamine induced a continuous catecholamine secretion (EC50= 3 × 10−7M) via the H1 receptor, in addition to an initial catecholamine burst due to a nonspecific stimulatory effect at higher doses (≥10−4M). The continuous secretion showed little desensitization and lasted for more than 1 h. In fura‐2‐loaded cells, the stimulation with histamine evoked a transient rise of intracellular free Ca2+ concentration ([Ca2+]i) which lasted only for a few minutes and was followed by a sustained [Ca2+]i rise which continued for more than 20 min. The addition of an activator for the L‐type voltage‐sensitive Ca2+ channel, i.e., Bay K 8644 (1 μM), facilitated the sustained [Ca2+]i rise, as well as the secretion, whereas the addition of relatively high concentrations of Ca2+‐channel blockers (10 μM) suppressed the sustained [Ca2+]i rise and part of the secretion. Removal of extracellular Ca2+ completely abolished continuous secretion and sustained [Ca2+]irise. When the external Ca2+ level was elevated, both sustained (Ca2]i rise and continuous secretion were enhanced in a similar Ca2+‐dependent manner, showing saturation with around 1–3 mM Ca2+. This Ca2+ dependence was clearly different from that observed with high K+ and nicotine, which is mediated by the L‐type Ca2+ channel, in which the responses showed little or no saturation when the Ca2+ level was increased. The results indicate that stimulation with histamine induces a continuous secretion via the H1 receptor, in addition to a transient and nonspecific secretion at higher doses. At least part of the continuous secretion is induced by a sustained [Ca2+]i rise due to the continuous Ca2+ influx through Ca2+ channels. These channels are sensitive to dihydropyridines, but do not appear to be identical to the L‐type voltage‐sensitive Ca2+ channel.
Somatostatin Release from Rat Cerebral Cortex Synaptosomes
Authors: Bonanno G. , Parodi B. , Cafaggi S. , Raiteri M. 
Published: October 2006

AbstractRat cerebral cortex synaptosomes were exposed in superfusion to various depolarizing stimuli and the release of somatostatin‐like immunoreactivity (SRIF‐LI) was measured by means of a radioimmunoassay procedure. High KC1 (9‐50 mM) concentration dependently evoked SRIF‐LI release; the evoked overflow reached a plateau at 25 mM KC1 and was completely abolished when Ca2+ ions were omitted from the superfusion medium, independently of the concentration of KC1 used. The 15 mM K+‐evoked release of SRIF‐ LI increased sharply as the Ca2+ concentration was raised to 0.8 mM, then leveled off and reached a plateau at 1.2 mM. The 15 mM K+‐evoked overflow, but not the spontaneous outflow, was partially decreased (50%) by 1 μM tetrodotoxin. The presence in the superfusion fluid of a mixture of peptidase inhibitors did not improve the recovery of SRIF‐LI both in the absence and in the presence of high K+. Exposure of synaptosomes to veratrine (1‐50 μM) induced release of SRIF‐LI in a concentration‐dependent way. The effect of the alkaloid was strictly Ca2+ and tetrodotoxin sensitive. Replacement of extracellular Na+ by sucrose caused an acceleration of the spontaneous SRIF‐LI outflow that was inversely correlated to the Na+ content in the superfusion medium. The release evoked by the sodium‐deprived media did not exhibit any calcium dependence. HPLC analysis of the samples collected during superfusion showed that >90% of the SRIF‐LI released either during the spontaneous outflow or by 15 mM KC1 was represented by SRIF‐14 (SRIF‐2814‐28). These values reflected the ratio SRIF‐14/SRIF‐28 found in synaptosomes at the end of the experiments.
Effects of Monensin and Veratridine on Acetylcholine Release and Cytosolic Free Ca2+ Levels in Cerebrocortical Synaptosomes of Rats
Authors: Satoh Eiki , Nakazato Yoshikazu 
Published: October 2006

AbstractMonensin (10−8‐10−4M) caused a dose‐dependent increase in the release of [3H] acetylcholine ([3H] ACh) from purified rat cerebrocortical synaptosomes, with an EC50 of ∼1.6 × 10−6M. Extracellular Na+, but not Ca2+, was required for a monensin‐induced increase in the release of [3H] ACh. Monensin also increased the cytosolic free Ca2+ concentration ([Ca2+]i) and uptake of 22Na+ in a dose‐dependent manner. Monensin continued to cause a dose‐dependent increase in [Ca2+]i in the absence of extracellular Ca2+, although an ∼50% reduction was noted at concentrations of >10−5M. The EC50 for the monensin‐induced increase in [Ca2+]i was similar to that noted in the release of [3H]ACh. Veratridine exhibited effects similar to those of monensin, but a large portion of the increase in [Ca2+]i and [3H] ACh release was dependent on extracellular Ca2+. Measurements of rhodamine 6G fluorescence indicated that monensin and veratridine caused synaptosomal hyperpolarization and depolarization, respectively. Tetrodotoxin (10−6M) completely blocked all the effects of veratridine but had no effect on the activity of monensin. These results suggest that monensin increases the release of ACh at least in part by increasing [Ca2+]i, resulting from the increase in the Na+ influx through tetrodotoxin‐insensitive mechanisms in rat cerebrocortical synaptosomes.
In Vivo Studies Identify 5a‐Pregnan‐3a‐o1‐20‐one as an Active Anesthetic Agent
Authors: Mok Wai Man , Herschkowitz Sylvia , Krieger Neil R. 
Published: October 2006

AbstractMice were anesthetized with 5a‐[3H]pregnan‐3a‐o1‐20‐one. Brain levels for 5α‐pregnan‐3α‐o1–20‐one and its five major metabolites (5α‐pregnanedione, ko, k1, k2, k3) were compared at behavioral endpoints that are characteristic of the anesthetized state. The results support the hypothesis that 5α‐pregnan‐3α‐o1–20‐one mediates the anesthetic response, and they weigh against the hypothesis that any of its metabolites is solely responsible for the onset or the maintenance of the anesthetized state. For an administered dose of 3 mg/kg, brain levels (means ± SEM) for 5α‐pregnan‐3α‐o1–20‐one at the time of the loss of the righting response (n = 10) and at the time of the return of the righting response (n = 6) were 7.24 ± 0.61 pmol/mg of brain tissue and 3.63 ± 0.26 pmol/mg of brain tissue, respectively. No metabolite level was lower at the return of the righting response than at the loss of the righting response. 5α‐Pregnan‐3′‐o1–20‐one brain levels increased consistently with the percentage of anesthetized mice. This was not the case for any of the metabolites. Fifty percent of the mice were anesthetized when the 5α‐pregnan‐3α‐o1–20‐one level was 4.5 pmol/mg of brain tissue.
Okadaic Acid‐Induced Inhibition of B‐50 Dephosphorylation by Presynaptic Membrane‐Associated Protein Phosphatases
Authors: Han Yi‐fan , Dokas Linda A. 
Published: October 2006

AbstractThe neuronal tissue‐specific protein kinase C (PKC) substrate B‐50 can be dephosphorylated by endogenous protein phosphatases (PPs) in synaptic plasma membranes (SPMs). The present study characterizes membrane‐associated B‐50 phosphatase activity by using okadaic acid (OA) and purified 32P‐labeled substrates. At a low concentration of [γ‐32P] ATP, PKC‐mediated [32P]phosphate incorporation into B‐50 in SPMs reached a maximal value at 30 s, followed by dephosphorylation. OA, added 30 s after the initiation of phosphorylation, partially prevented the dephosphorylation of B‐50 at 2 nM, a dose that inhibits PP‐2A. At the higher concentration of 1 μM, a dose of OA that inhibits PP‐1 as well as PP‐2A, a nearly complete blockade of B‐50 dephosphorylation was seen. Heat‐stable PP inhibitor‐2 (1–2) also inhibited dephosphorylation of B‐50. The effects of OA and 1–2 on B‐50 phosphatase activity were additive. Endogenous PP‐1‐ and PP‐2A‐like activities in SPMs were also demonstrated by their capabilities of dephosphorylating [32P] phosphorylase a and [32P] casein. With these exogenous substrates, sensitivities of the membrane‐bound phosphatases to OA and 1–2 were found to be similar to those of purified forms of these enzymes. These results indicate that PP‐1‐ and PP‐2A‐like enzymes are the major B‐50 phosphatases in SPMs.