bluuebunny/crossref_metadata_2025 huggingface dataset:
Effect of Unilateral Perinatal Hypoxic‐Ischemic Brain Injury in the Rat on Dopamine D1 and D2 Receptors and Uptake Sites: A Quantitative Autoradiographic Study
Authors: Przedborski Serge , Kostic Vladimir , Jackson‐Lewis Vernice , Cadet Jean Lud , Burke Robert E.
Published: October 2006

The effect of a unilateral perinatal hypoxic‐ischemic brain injury on dopamine D1 and D2 receptors and uptake sites was investigated in rats by using in vitro quantitative binding autoradiography, 2–3 weeks after the insult. We observed significant decreases in the Bmax and KD for [3H]SCH 23390‐labeled D1 and in the Bmax for [3H]spiperone‐labeled D2 receptors in the lesioned caudate‐putamen in rats with moderate brain injury (visible loss in hemispheric volume ipsilateral to the injury) compared with the nonlesioned contralateral caudate‐putamen or with control rats. Changes in [3H]SCH 23390 and [3H]spiperone binding predominated in the dorsolateral part of the lesioned caudate‐putamen. Pronounced reduction in [3H]SCH 23390 binding was also observed in the substantia nigra pars reticulata on the side of the lesion. In contrast, we did not observe any significant change in Bmax or KD for [3H]mazindol‐labeled dopamine uptake sites. Similarly, no significant changes in the levels of dopamine or its metabolites were found on the side of the lesion. The observed reductions in striatal dopamine D1 and D2 receptors are a reflection of striatal cell loss induced by the hypoxic‐ischemic injury. The absence of changes in [3H]mazindoI binding or dopamine levels in the lesioned caudate‐putamen indicates that the dopaminergic presynaptic structures are preserved.
Isolation and Characterization of Rapid Transport Vesicle Subtypes from Rabbit Optic Nerve
Authors: Morin Peter J. , Liu Ningai , Johnson Robin J. , Leeman Susan E. , Fine Richard E.
Published: October 2006

Abstract: Subcellular fractionation of rabbit optic nerve resolves three populations of membranes that are rapidly labelled in the axon. The lightest membranes are >200 nm and are relatively immobile. The intermediate density membranes consist of 84 nm vesicles which disappear from the nerve with kinetics identical to those of the rapid component. A third population of membranes, displaying a distinct protein profile, is present in the most dense region of the gradient. Immunological characterization of these membranes suggests the following. (1) The lightest peak contains rapidly transported glucose transporter and most of the total glucose transporters present in the nerve; this peak is therefore enriched in axolemma. (2) The intermediate peak contains rapidly transported glucose transporters and synaptophysin, an integral synaptic vesicle protein, and about half of the total synaptophysin; this peak therefore contains transport vesicles bound for both the axolemma and the nerve terminal, and these subpopulations can be separated by immunoadsorption with specific antibodies against the aforementioned proteins. (3) The heaviest peak contains rapidly transported synaptophysin and tachykinin neuromodulators and about half of the total synaptophysin, and 80% of the total tachykinins present in the nerve; this peak appears to represent a class of synaptic vesicle precursor bound for the nerve terminal exclusively. (4) Synaptophysin is present in the membranes of vesicles carrying tachykinins. (5) Both the intermediate and the heaviest peaks are enriched in kinesin heavy chain, suggesting that both vesicle classes may be transported by the same mechanism.
65Zn Uptake from Blood into Brain in the Rat
Authors: Pullen R. G. L. , Franklin P. A. , Hall G. H.
Published: October 2006

Abstract: Zinc is essential for the normal development and function of the CNS, although little is known about brain zinc homeostasis. Therefore, in this investigation we have studied 65Zn uptake by brain from blood and have measured the blood‐brain barrier permeability to 65Zn in the anaesthetised rat in vivo. Adult male Wistar rats within the weight range 500–600 g were used. 65ZnCl2 and 125I‐albumin, the latter serving as a vascular marker, were injected intravenously in a bolus of normal saline. Sequential arterial blood samples were taken during experiments that lasted between 5 min and 5 h, after which the whole brain was removed, dissected, and analysed for radioisotope activity. Data have been analysed by graphical analysis, which suggests that after 30 min of circulation, 65Zn uptake by brain from blood is unidirectional with an influx rate constant, Kin, of ∼5 × 10−4 ml/min/g. At circulation times of <30 min, 65Zn fluxes between blood and brain are bidirectional, where influx has a K value of >5 × 10−4 ml/min/g. In addition to the blood space, the brain appears to contain a rapidly exchanging compartment(s) for 65Zn of ∼4 ml/100 g, which is not CSF.
Astrocytes, Not Neurons, Produce Docosahexaenoic Acid (22:6ω‐3) and Arachidonic Acid (20:4ω‐6)
Authors: Moore Steven A. , Yoder Elizabeth , Murphy Sean , Dutton Gary R. , Spector Arthur A.
Published: October 2006

Abstract: Elongated, highly polyunsaturated derivatives of linoleic acid (18:2ω‐6) and linolenic acid (18:3ω‐3) accumulate in brain, but their sites of synthesis are not fully characterized. To investigate whether neurons themselves are capable of essential fatty acid elongation and desaturation or are dependent upon the support of other brain cells, primary cultures of rat neurons and astrocytes were incubated with [1‐14C]18:2ω‐6, [1‐14C]20:4ω‐6, [1‐14C]18:3ω‐3, or [1‐14C]20:5ω‐3 and their elongation/desaturation products determined. Neuronal cultures were routinely incapable of producing significant amounts of Δ4‐desaturase products. They desaturated fatty acids very poorly at every step of the pathway, producing primarily elongation products of the 18‐ and 20‐carbon precursors. In contrast, astrocytes actively elongated and desaturated the 18‐ and 20‐carbon precursors. The major metabolite of 18:2ω‐6 was 20:4ω‐6, whereas the primary products from 18:3ω‐3 were 20:5ω‐3, 22:5ω‐3, and 22:6ω‐3. The majority of the long‐chain fatty acids formed by astrocyte cultures, particularly 20:4ω‐6 and 22:6ω‐3, was released into the extracellular fluid. Although incapable of producing 20:4ω‐6 and 22:6ω‐3 from precursor fatty acids, neuronal cultures readily took up these fatty acids from the medium. These findings suggest that astrocytes play an important supportive role in the brain by elongating and desaturating ω‐6 and ω‐3 essential fatty acid precursors to 20:4ω‐6 and 22:6ω‐3, then releasing the long‐chain polyunsaturated fatty acids for uptake by neurons.
Influence of Bradykinin on Diacylglycerol and Phosphatidic Acid Accumulation in Cultured Bovine Adrenal Chromaffin Cells
Authors: Owen P. Jane , Boarder Michael R.
Published: October 2006

Earlier studies have shown that bradykinin stimulated release of catecholamines from chromaffin cells by an influx of calcium through dihydropyridine‐insensitive channels, and also that bradykinin stimulated (poly)phos‐ phoinositide hydrolysis. To investigate membrane‐bound second messengers in chromaffin cells, and to elucidate any role these may play in stimulus‐secretion coupling, we have studied the influence of bradykinin on diacylglycerol and phosphatidic acid (PA). Using equilibrium labelling of primary cultures of chromaffin cells with [3H]arachidonic acid or [3H]glycerol, we found no influence of bradykinin (10 nM) on labelled diacylglycerol formation, either in the presence or absence of inhibitors of diacylglycerol lipase or kinase. However, when we used cells prelabelled with 32Pi for 2.5 h we found that bradykinin produced a substantial stimulation of label found in PA, with an EC50 value of about 1 nM. This bradykinin stimulation of [32P]PA formation was only partially dependent on extracellular calcium, in contrast to the smaller response to nicotine, which was completely dependent on extracellular calcium. Short (10 min) pretreatment with tetradecanoylphorbol acetate (TPA) almost completely eliminated the bradykinin‐stimulated formation of inositol phosphates, but failed to affect bradykinin stimulation of label in PA, suggesting that PA production in response to bradykinin is not downstream of phospholipase C activation. TPA alone failed to stimulate [32P]PA substantially, whereas long‐term (24 or 48 h) treatment with TPA failed to attenuate the response to bradykinin. Diacylglycerol kinase inhibitors were also without effect on the bradykinin stimulation of [32P]PA. These results suggest that bradykinin stimulates PA production by a mechanism independent of the activation of protein kinase C. A preliminary indication that G proteins may be involved was suggested by the observation that AIF4 stimulates [32P]PA accumulation whereas N‐ethylmaleimide inhibits bradykinin‐stimulated [32P]PA accumulation.
Noradrenaline Release from Streptolysin O‐Permeated Rat Cortical Synaptosomes: Effects of Calcium, Phorbol Esters, Protein Kinase Inhibitors, and Antibodies to the Neuron‐Specific Protein Kinase C Substrate B‐50 (GAP‐43)
Authors: Dekker Lodewijk V. , De Graan Pierre N. E. , Pijnappel Pim , Oestreicher A. Beate , Gispen Willem Hendrik
Published: October 2006

Abstract: We studied the molecular mechanism of noradrenaline release from the presynaptic terminal and the involvement of the protein kinase C substrate B‐50 (GAP‐43) in this process. To gain access to the interior of the presynaptic terminal, we searched for conditions to permeate rat brain synaptosomes by the bacterial toxin streptolysin O. A crude synaptosomal/mitochondrial preparation was preloaded with [3H]noradrenaline. After permeation with 0.8 IU/ml streptolysin O, noradrenaline efflux could be induced in a concentration‐dependent manner by elevating the free Ca2+ concentration from 10−8 to 10−5M. Efflux of the cytosolic marker protein lactate dehydrogenase was not affected by this increase in Ca2+. Ca2+‐induced efflux of noradrenaline was largely dependent on the presence of exogenous ATP. Changing the Na+/K+ ratio in the buffer did not affect Ca2+‐induced noradrenaline release. Release of noradrenaline could also be evoked by phorbol esters, indicating the involvement of protein kinase C. Ca2+‐ and phorbol ester‐induced release were not additive at higher phorbol ester concentrations (>10−7M). We compared the sensitivities of Ca2+‐ and phorbol ester‐induced release of noradrenaline to the protein kinase inhibitors H‐7 and polymyxin B and to antibodies raised against synaptic protein kinase C substrate B‐50. Ca2+‐induced release was inhibited by B‐50 antibodies and polymyxin B, but not by H‐7; phorbol ester‐induced release was inhibited by polymyxin B and by H‐7, but only marginally by antibodies to B‐50. We suggest that phorbol esters and Ca2+ stimulate noradrenaline release through different mechanisms and that the essential role of B‐50 in Ca2+‐induced noradrenaline release may involve other properties of B‐50 besides protein kinase C–mediated phosphorylation.
Negative Effects of Tacrine (Tetrahydroaminoacridine) and Methoxytacrine on the Metabolism of Acetylcholine in Brain Slices Incubated Under Conditions Stimulating Neurotransmitter Release
Authors: Tuček Stanislav , Doležal Vladimír
Published: October 2006

Abstract: The effects of tacrine (1,2,3,4‐tetrahydro‐9‐aminoacridine) and 7‐methoxytacrine on the metabolism of brain acetylcholine were investigated in experiments in which acetylcholine turnover was stimulated by tissue depolarization or by 4‐aminopyridine. Rat cerebrocortical prisms were preincubated under “resting”conditions (Krebs‐Ringer buffer with 3 mmol/L K+ and with paraoxon to inhibit cholinesterases) and then incubated in the presence of tacrine or methoxytacrine and of 50 mmol/L K+. Both drugs diminished the amount of acetylcholine released by depolarization and the amount of acetylcholine synthesized during incubation; in experiments in which [14]choline was present in the incubation medium simultaneously with tacrine or methoxytacrine, the drugs diminished the uptake of [14C]choline by the tissue and the amount of [14C]‐acetylcholine synthesized and released into the medium. In these experiments, it was not possible to distinguish whether the inhibitory effects of tacrine and methoxytacrine were primarily on the process of acetylcholine synthesis (particularly on the uptake of choline), or whether the drugs also acted directly on the process of neurotransmitter release. In subsequent experiments the prisms were preincubated with [14C]choline and only then subjected to a short depolarization in the presence of hemicholinium‐3 and tacrine or methoxytacrine. Both drugs severely inhibited the release of preformed [14C]acetylcholine and prevented the diminution of tissue [14C]acetylcholine stores. Methoxytacrine was also found to diminish the release of acetylcholine induced by 4‐aminopyridine while increasing the content of acetylcholine in the tissue. Tacrine and methoxytacrine had no effect on the activity of choline acetyltransferase (EC 2.3.1.6). Our observations indicate that, in addition to reducing the uptake of choline, tacrine and methoxytacrine inhibit the stimulated release of acetylcholine by acting directly on the process of neurotransmitter liberation. Most effects of tacrine and methoxytacrine were observed at a concentration of 100 μmol/L, but some were already apparent at a concentration of 10 μmol/L. The molecular mechanism of the direct inhibitory action of tacrine and methoxytacrine on the stimulated release of acetylcholine is not clear.
Endogenous and Expressed Angiotensin II Receptors on Xenopus Oocytes
Authors: Fluharty Steven J. , Reagan Lawrence P. , White Michael M.
Published: October 2006

Abstract: Intact Xenopus oocytes contain a homogeneous population of binding sites for the angiotensin II (Ang II) receptor antagonist 125I‐[Sarc1,Ile8]‐Ang II (125I‐SARILE). Binding of 125I‐SARILE to intact oocytes was saturable and of high affinity with an apparent KD of 0.7 nM and maximal density of 0.12 fmol/oocyte. Binding of 125I‐SARILE to oocytes also was specific for Ang II‐related peptides with a rank order potency of: [Sarc1]‐Ang II > Ang II > Ang III Ang I. However, these endogenous binding sites were present only in follicle‐enclosed oocytes and within the follicular layer itself. On the other hand, injection of poly(A)+ RNA isolated from murine N1E‐115 neuroblastoma cells into oocytes resulted in the appearance of 125I‐SARILE binding sites even in defolliculated oocytes. These expressed receptors exhibited pharmacological properties similar to those endogenously present in the follicular layer, although their levels were much less. Collectively, these results suggest that endogenous Ang II receptors are present on Xenopus oocyte follicle cells, whereas Ang II receptors expressed from exogenous N1E‐115 RNA are found on the oocytes themselves. In addition, the high density of Ang II receptors on the follicle cells emphasizes the necessity for care in using Xenopus oocytes for the expression of receptors encoded by exogenous RNAs.
Dihydroxyphenylalanine and Dopamine Are Released from Portal Vein Together with Noradrenaline and Dihydroxyphenylglycol During Nerve Stimulation
Authors: Hunter Larry W. , Rorie Duane K. , Tyce Gertrude M.
Published: October 2006

Abstract: The overflows of 3,4‐dihydroxyphenylalanine, dopamine, noradrenaline, and 3,4‐dihydroxyphenylglycol in canine portal vein superfused in vitro were studied before, during, and after depolarization of sympathetic nerve endings. The four compounds were separated from superfusate and from tissue on Sep‐Pak C‐18 cartridges and quantified by HPLC with electrochemical detection. Physiological and biochemical methods were used to show that the compound released was most probably 3,4‐dihydroxyphenylalanine; the identity of the other endogenous compounds has been established previously. Release of 3,4‐dihydroxyphenylalanine was calcium and frequency dependent, inhibited by α‐m‐L‐p‐tyrosine (an inhibitor of tyrosine hydroxylase) and augmented by 3‐hydroxybenzylhydrazine (an inhibitor of aromatic amino acid decarboxylase). The overflows of dopamine, noradrenaline, and 3,4‐dihydroxy phenylglycol from the vein were calcium and frequency dependent. It was estimated that under control conditions, ∼ 80% of the total 3,4‐dihydroxyphenylalanine that was synthesized was directed to catecholamine biosynthesis, ∼8% overflowed from the vein, and ∼ 14% remained unchanged within the tissue. It is concluded that 3,4‐dihydroxyphenylalanine and dopamine are released together with noradrenaline and 3,4‐dihydroxyphenylglycol from portal vein upon nerve depolarization.
The Binding Properties of the Particulate and Solubilized Sulfonylurea Receptor from Cerebral Cortex Are Modulated by the Mg2+ Complex of ATP
Authors: Schwanstecher Mathias , Schaupp Ursula , Löser Stefan , Panten Uwe
Published: October 2006

Abstract: Glibenclamide closes an ATP‐sensitive K+ channel (K‐ATP channel) by interaction with the sulfonylurea receptor in the plasma membrane of pancreatic B cells and thereby initiates insulin release. Previous studies demonstrated that the Mg2+ complex of ATP decreases glibenclamide binding to the sulfonylurea receptor from pancreatic islets. The aim of the present study was to examine the effect of adenine and guanine nucleotides on binding of sulfonylureas to the cerebral sulfonylurea receptor. For this purpose, binding properties of the particulate and solubilized site from rat or pig cerebral cortex were analyzed. Maximum recovery of receptors in detergent extracts amounted to 40–50%. Specific binding of [3H]glibenclamide to the solubilized receptors corresponded well to specific binding to microsomes. In microsomes and detergent extracts, the Mg2+ complexes of ATP, ADP, GTP, and GDP inhibited binding of [3H]glibenclamide. These effects were not observed in the absence of Mg2+. In detergent extracts, Mg‐ATP (300 μM) reduced the number of high‐affinity sites for [3H]‐glibenclamide by 52% and increased the dissociation constant for [3H]glibenclamide by eightfold; Mg‐ATP was half‐maximally effective at 41 μM. Alkaline phosphatase accelerated the reversal of Mg‐ATP‐induced inhibition of [3H]glibenclamide binding. The data suggest similar control of the sulfonylurea receptor from brain and pancreatic islets by protein phosphorylation.
Heterogeneity of [3H]Dipyridamole Binding to CNS Membranes: Correlation with [3H]Nitrobenzylthioinosine Binding and [3H]Uridine Influx Studies
Authors: Jones K. W. , Hammond J. R.
Published: October 2006

Abstract: The relationship between the nucleoside transport system and the nitrobenzylthioinosine‐sensitive and ‐resistant [3H]dipyridamole binding sites was examined by comparing the characteristics of [3H]dipyridamole binding with those of [3H]nitrobenzylthioinosine binding and [3H]‐uridine influx in rabbit and guinea pig cerebral cortical synaptosomes. Two distinct high‐affinity synaptosomal membrane‐associated [3H]dipyridamole binding sites, with different sensitivities to inhibition by nitrobenzylthioinosine, were characterized in the presence of 3–[(3‐cholamidopropyl)dimethylammonio]‐1 ‐propanesulfonate (CHAPS, 0.01%) to prevent [3H]dipyridamole binding to glass tubes and niters. The nitrobenzylthioinosine‐resistant [3H]‐dipyridamole binding sites represented a greater proportion of the total membrane sites in guinea pig than in rabbit (40 vs. 10% based on inhibition studies). In rabbit, nitrobenzylthioinosine‐sensitive [3H]dipyridamole binding (KD= 1.4 ± 0.2 nM) and [3H]nitrobenzylthioinosine binding (KD= 0.30 ± 0.01 nM) appeared to involve the same membrane site associated with the nitrobenzylthioinosine‐sensitive nucleoside transporter. By mass law analysis, [3H]‐dipyridamole binding in guinea pig could be resolved into two components based on sensitivity to inhibition by 1 μM nitrobenzylthioinosine. The nitrobenzylthioinosine‐resistant [3H]dipyridamole binding sites were relatively insensitive to inhibition by all of the nucleoside transport substrates and inhibitors tested, with the exception of dipyridamole itself. In guinea pig synaptosomes, 100 μM dilazep blocked nitrobenzylthioinosine‐resistant [3H]uridine transport completely but inhibited the nitrobenzylthioinosine‐resistant [3H]dipyridamole binding component by only 20%. Furthermore, a greater percentage of the [3H]‐dipyridamole binding was nitrobenzylthioinosine resistant in guinea pig compared with rabbit, yet both species had a similar percentage of nitrobenzylthioinosine‐resistant [3H]‐uridine transport. These data suggest that the nitrobenzylthioinosine‐resistant [3H]dipyridamole binding site(s) involves membrane components distinct from those associated with functional, nitrobenzylthioinosine‐resistant, nucleoside transporters.
Alterations in Extracellular and Tissue Levels of Biogenic Amines in Rat Brain Induced by the Serotonin2 Receptor Antagonist, Ritanserin
Authors: Devaud Leslie L. , Hollingsworth Elizabeth B. , Cooper Barrett R.
Published: October 2006

Abstract: Systemic administration of ritanserin elicited rapid changes in dopamine (DA) and serotonin (5‐HT) levels in both dialysate and neuronal tissue extracts. These effects occurred in both a site‐selective and a dose‐related manner. Increases in extracellular levels of DA and 5‐HT in the nucleus accumbens were maximal at 120–140 min after treatment. A dose of 0.63 mgJ.kg of ritanserin elicited larger and more prolonged increases in extracellular DA and 5‐HT levels than did the 0.3 mgJ.kg dose. By contrast, 0.63 mgJ.kg of ritanserin elicited no changes in either DA or 5‐HT levels with dialysate collected from the striatum. Ritanserin also induced dose‐related decreases in tissue levels of DA and 5‐HT from the nucleus accumbens. The site specificity of action was again noted in that there were no dose‐dependent decreases in tissue levels of DA or 5‐HT measured from the striatum. Ritanserin exerted little effect on metabolite levels from either dialysate or tissue extracts. Taken together, these findings show that selective 5‐HT2 receptor antagonism modulates DA and 5‐HT neurotrans‐mission in a specific manner. These actions appear to involve increased release of DA and 5‐HT rather than significant changes in metabolism. These findings add further weight to the importance of 5‐HT2 receptor interactions as an important component of antipsychotic activity.
Time‐Dependent Inhibition of Stimulatéd Adrenal Catecholamine Release by Staurosporine
Authors: Maurer Jennifer A. , McKay Dennis B.
Published: October 2006

Abstract: Staurosporine, a potent inhibitor of protein kinases, is used to study the involvement of protein kinases in cellular processes. In the present studies, the effect of prolonged staurosporine treatment on catecholamine secretion in cultured bovine adrenal chromaffin cells was examined. Staurosporine inhibits catecholamine release stimulated by 10 μM nicotine, depolarizing concentrations of potassium (56 mM KCl), and 2 mM BaCl2. The effects of staurosporine on KCl‐stimulated release are time dependent, with a half‐time of ∼50 min and a maximal inhibition at 2 h. Our results indicate that activation of a staurosporine‐sensitive protein kinase is not directly involved in the stimulus‐secretion coupling process. This does not rule out the possibility that Ca2+/phospholipid‐dependent protein kinase or other protein kinases may acutely modulate release. However, these results suggest that a protein(s), which is phosphorylated by a staurosporine‐sensitive protein kinase(s), is required to maintain the integrity of the stimulus‐secretion coupling process.
Protein Phosphorylation in Bovine Adrenal Medullary Chromaffin Cells: Histamine‐Stimulated Phosphorylation of Tyrosine Hydroxylase
Authors: Bunn S. J. , Harrison S. M. , Dunkley P. R.
Published: October 2006

Histamine can cause the release of catechol‐amines from bovine adrenal medullary chromaffin cells by a mechanism distinct from that of the depolarizing agents nicotine or high K+ buffer. It was the aim of this study to determine the protein phosphorylation responses to histamine in these cells and to compare them with those induced by depolarization. A number of proteins showed increases in phosphorylation in response to histamine especially when analyzed on two‐dimensional polyacrylamide gel elec‐trophoresis or by phosphopeptide mapping; one protein of 20,000 daltons was markedly dephosphorylated. Emphasis was given to the effects of histamine on tyrosine hydroxylase (TOH) phosphorylation, because this protein showed the most prominent changes on one‐dimensional gels. Histamine acted via H1 receptors to increase TOH phosphorylation; the response was blocked by the H1 antagonist mepyr‐amine and could be mimicked by the H1 agonist thiazolyl‐ethylamine, but not by the H2 agonist dimaprit. The H3 agonist (R)α‐methylhistamine increased TOH phosphorylation at high concentrations, but the response was blocked entirely by mepyramine. Histamine rapidly increased the phosphorylation of TOH, with a maximum reached within 5s and maintained for at least 30 min. This was in marked contrast to nicotine‐stimulated protein phosphorylation of TOH, which was rapidly desensitized. The initial phosphorylation response to histamine was independent of extracellular Ca2+ for at least 3 min, but the sustained response required extracellular Ca2+. This was in contrast to the situation with both nicotine and high K+ buffer, which under the conditions used here caused a response which was dependent on extracellular Ca2+ at all times investigated. In the presence of histamine, the phosphopeptide profiles for TOH were essentially the same with or without Ca2+, suggesting that the same protein kinases were involved, but at longer times there was evidence of new phosphorylation sites. The mechanism or mechanisms whereby histamine modulates TOH phosphorylation are discussed with emphasis on the differences from depolarizing agents.
Forskolin Stimulation of Cyclic AMP Accumulation in Rat Brain Cortex Slices Is Markedly Enhanced by Endogenous Adenosine
Authors: DeLapp Neil W. , Eckols Kris
Published: October 2006

Abstract: Stimulation of cyclic AMP (cAMP) accumulation in rat cortex slices by 1 μM forskolin (F) was markedly reduced (96%) by treatment with adenosine deaminase (ADA). The effect of ADA was progressively less at higher concentrations of F, but still inhibited the response by 50% at 100 μM F. ADA‐mediated inhibition of the cAMP response to 1 μM F was completely reversed by 5 μM 2‐chloroadenosine (CA), an ADA‐resistant analogue. Stimulation by F (controls) and F plus CA (ADA treated) in cortex slices was significantly inhibited by 200 μM caffeine (CAF) and by 10 μM 8‐phenyltheophylline. cAMP accumulation in ADA‐treated cortex slices stimulated with CA at concentrations from 5 to 100 μM was markedly enhanced by 1 μM F. Neither ADA treatment nor 200 μM CAF significantly affected cAMP accumulation in slices stimulated by 1 μM vasoactive intestinal polypeptide or adenylate cyclase in membranes stimulated by 1 μM F. CAF (1 mM) did not significantly increase basal cAMP levels in cortex slices, whereas 1 mM 3‐isobutyl‐1‐methylxanthine caused a significant 80% increase and 100 μM rolipram enhanced cAMP levels by 4.5‐fold. F‐stimulated cAMP accumulation (1 μM) in cortex slices was inhibited 98% by 1 mM CAF and 49% by 1 mM 3‐isobutyl‐1‐methylxanthine, and was enhanced 2.5‐fold by 100 μM rolipram. These data have been interpreted to indicate that the stimulation of cAMP accumulation in rat cortex slices by 1 μM F is predominantly due to synergistic interaction with endogenous adenosine and that the inhibition of this response by CAF is largely due to blockade of adenosine receptors.
Design of an Affinity Matrix for Purification of the Histamine H1 Receptor from Guinea Pig Cerebellum
Authors: Ruat M. , Traiffort E. , Garbarg M. , Schwartz J. C. , Demonchaux P. , Ife R. J. , Tertiuk W. , Ganellin C. R.
Published: October 2006

Abstract: H1 receptors from guinea pig cerebellum were solubilized using digitonin, and [125I]iodobolpyramine was used as a probe. [125I]Iodobolpyramine binding to this solubilized preparation occurred with a KD of 0.1 nM and a Bmax of 220 fmol/mg of protein and was inhibited by various H1 ligands with the expected potencies. Using a gel filtration procedure, a very sensitive radioassay was set up for detecting H1 activity in the solubilized preparation: 0.1 nM[125I]iodobolpyramine specific binding represented >90% of total binding. Moreover, the synthesis is described of potent H1 antagonists that are mepyramine derivatives with an amino alkyl acylamido alkyl spacer arm. One of them, UCL 1057 (Ki= 0.5 nM), has been coupled to a Sepharose epoxy‐activated resin. The resulting affinity matrix adsorbed selectively [125I]iodobolpyramine binding sites from the guinea pig cerebellum soluble preparation. In contrast, a Sepharose–glycine matrix was not able to adsorb these sites.
Differential Metabolism of Hydroxyeicosatetraenoic Acid Isomers by Mouse Cerebromicrovascular Endothelium
Authors: Giordano Michael J. , Mathur Satya N. , Moore Steven A.
Published: October 2006

Abstract: Hydroxyeicosatetraenoic acid (HETE) derivatives of arachidonic acid are produced in the brain and have been implicated as pathologic mediators in various types of brain injury. To understand better their fate in the brain, particularly in cerebral microvessels, several HETEs were incubated with cultured mouse cerebromicrovascular endothelium for 1, 2, and 4 h, followed by HPLC analysis of medium and cellular lipids. 5(S)‐ 8(RS)‐, and 9(RS)‐HETE were not metabolized by the cells, but were extensively incorporated, unmodified, into cell lipids. On the other hand, 11(RS)‐, 12(S)‐, and 15(S)HETE were extensively metabolized and only minimally incorporated into cell lipids. Previously, the major 12‐HETE metabolite was identified as 8‐hydroxyhexadecatrienoic acid. In the present study, we identified the major 11‐HETE metabolite as 7‐hydroxyhexadecatrienoic acid and the major 15‐HETE metabolite as 11‐hydroxyhexadecatrienoic acid. ω‐3 compounds, 15(S)‐ and (S)‐hydroxyeico‐sapentaenoic acids (HEPE), were also metabolized to more polar compounds, but to a lesser extent than their tetraenoic acid, ω‐6 counterparts. Comparison of 5‐, 12‐, and 15‐HETE enantiomers revealed no differences in metabolism or incorporation between the R and S stereoisomers. These data suggest that many isomers of HETE and HEPE can be incorporated into cell lipids or metabolized by pathways that do not distinguish between enantiomers. These pathways, however, are sensitive to the position or number of double bonds and are selective based on the position of the hydroxyl group.
Alzheimer β/A4‐Amyloid Precursor Protein: Evidence for Putative Amyloidogenic Fragment
Authors: Gandy Samuel E. , Bhasin Ramaninder , Ramabhadran Triprayar V. , Koo Edward H. , Price Donald L. , Goldgaber Dmitry , Greengard Paul
Published: October 2006

Abstract: Recombinant baculovirus was used to overexpress human Alzheimer β/A4‐amyloid precursor protein (APP) in Spodoptera frugiperda (Sf9) cells. Lysates of these cells were then analyzed for the presence of carboxyl‐terminal fragments of APP by an immuno‐blotting assay using either an antibody against the APP cytoplasmic domain (rabbit anti‐human 695APP645–694 or an antibody against the amino terminus of β/A4‐amyloid (rabbit anti‐human 695APP586–606). Anti‐human 695APP645–694 identified APP holoprotein, a 25‐kDa species, and a prominent group of carboxyl‐terminal fragments of 17, 16, and 14 kDa, whereas anti‐human 695APP586–606 identified APP holoprotein and a single prominent low‐molecular‐mass protein species comigrating with the 17‐kDa carboxyl‐terminal fragment identified by anti‐human 695APP645–694. No immunoreactive species was detected at these molecular mass positions when either antibody was used for analysis of lysates of either uninfected Sf 9 cells or Sf 9 cells infected with wild‐type Autographa californica baculovirus. For each antibody, specific immunoreactivity was abolished by preabsorption with the corresponding peptide immunogen. The incorporation of a β/A4‐amyloid amino‐terminal epitope into a 17‐kDa fragment of APP suggests that, in the baculoviral overexpression system, the electrophoretic microheterogeneity of APP carboxyl‐terminal fragments is due, at least in part, to alternative proteolysis of APP. If such carboxyl‐terminal fragments of APP containing an intact β/A4‐amyloid domain are produced in human brain, then they may represent intermediates in the conversion of APP to deposited β/A4‐amyloid. The identification of potentially amyloidogenic fragments in recom‐binantly engineered Sf 9 cells may provide a useful experimental system for determination of alternative sites of APP proteolysis and investigation of the processing mechanisms involved.
Quantitative Autoradiography of Dihydrorotenone Binding to Complex I of the Electron Transport Chain
Authors: Greenamyre J. Timothy , Higgins Donald S. , Eller Robert V.
Published: October 2006

Abstract: Defective complex I activity has been linked to Parkinson's disease and Huntington's disease, but little is known of the regional distribution of this enzyme in the brain. We have developed a quantitative autoradiographic assay using [3H]dihydrorotenone ([3H]DHR) to label and localize complex I in brain tissue sections. Binding was specific and saturable and in the cerebellar molecular layer had a KD of 11.5 ± 1.3 nM and a Bmax of 11.0 ± 0.4 nCi/mg of tissue. Unlabeled rotenone and 1‐methyl‐4‐phenylpyridinium ion competed effectively for DHR binding sites. Binding was markedly enhanced by 100 μM NADH. The distribution of complex 1 in brain, as revealed by DHR autoradiography, is unique but somewhat similar to that of cytochrome oxidase (complex IV). This assay may provide new insight into the roles of complex I in brain function and neurodegeneration.
C‐Type Natriuretic Peptide Is a Potent Stimulator of Cyclic GMP Production in Cultured Mouse Astrocytes
Authors: Yeung V. T. F. , Ho S. K. S. , Cockram C. S. , Lee C. M. , Nicholls M. G.
Published: October 2006

Abstract: The effect of C‐type natriuretic peptide (CNP), a novel member of the natriuretic peptide family, on cyclic GMP (cGMP) generation was studied in primary cultures of mouse astrocytes. CNP stimulated cGMP production by mouse astrocytes in a dosedependent fashion, with an EC50 of 32 nM and a maximal stimulatory concentration of > 1 μM, which induced a rise of cGMP level from a baseline of 1.0 ± 0.1 pmol/mg of protein to 196.2 ± 22.0 pmol/mg of protein. Compared with our previously reported atrial and brain natriuretic peptide‐induced cGMP responses, CNP had a lower EC50 and was 10‐20 times more efficacious in its maximal effect on cGMP stimulation. These data lend support to the concept of a significant role of CNP in neuromodulation/neurotransmission.
Muscarinic Autoreceptors of Torpedo Electric Organ Are of the M1 Subtype: Evidence by Radioligand Binding Using Selective Antagonists
Authors: Green A. C. , Dowdall M. J.
Published: October 2006

Abstract: The presynaptic muscarinic autoreceptor of Torpedo marmorata electric organ has been characterised by radioligand binding studies using the subtype‐selective antagonists pirenzepine, (+)‐telenzepine, methoctramine, and AF‐DX 116. The presynaptic receptor had relatively high affinity for the M1 antagonists pirenzepine and (+)‐telenzepine (Ki= 35 and 7 nM, respectively) and lower affinities for the M2 antagonists AF‐DX 116 and methoctramine (Ki= 311 and 277 nM, respectively). Comparison of these binding data with those from an M2 receptor (rat heart membranes) assayed under identical conditions and with data in the recent literature suggests that the Torpedo muscarinic autoreceptor has a pharmacology most similar to the M1 pharmacological subtype of muscarinic acetylcholine receptor.
Development and Maintenance of the Neuronal Cytoskeleton in Aggregated Cell Cultures of Fetal Rat Telencephalon and Influence of Elevated K+ Concentrations
Authors: Riederer B. M. , Monnet‐Tschudi F. , Honegger P.
Published: October 2006

Abstract: Serum‐free aggregating cell cultures of fetal rat telencephalon were examined by biochemical and immunocytochemical methods for their development‐dependent expression of several cytoskeletal proteins, including the heavy‐ and medium‐sized neurofilament subunits (H‐NF and M‐NF, respectively); brain spectrin; synapsin I; β‐tubulin; and the microtubule‐associated proteins (MAPs) 1, 2, and 5 and τ protein. It was found that with time in culture the levels of most of these cytoskeletal proteins increased greatly, with the exceptions of the particular β‐tubulin form studied, which remained unchanged, and MAP 5, which greatly decreased. Among the neurofilament proteins, expression of M‐NF preceded that of H‐NF, with the latter being detectable only after ∼ 3 weeks in culture. Furthermore, MAP 2 and τ protein showed a development‐dependent change in expression from the juvenile toward the adult form. The comparison of these developmental changes in cytoskeletal protein levels with those observed in rat brain tissue revealed that protein expression in aggregate cultures is nearly identical to that in vivo during maturation of the neuronal cytoskeleton. Aggregate cultures deprived of glial cells, i.e., neuron‐enriched cultures prepared by treating early cultures with the antimitotic drug cytosine arabinoside, exhibited pronounced deficits in M‐NF, H‐NF, MAP 2, MAP 1, synapsin I, and brain spectrin, with increased levels of a 145‐kDa brain spectrin breakdown product. These adverse effects of glial cell deprivation could be reversed by the maintenance of neuron‐enriched cultures at elevated concentrations of KCl (30 mM). This chronic treatment had to be started at an early developmental stage to be effective, a finding suggesting that sustained depolarization by KCl is able to enhance the developmental expression and maturation of the neuronal cytoskeleton.
Barium and Calcium Stimulate Secretion from Digitonin‐Permeabilized Bovine Adrenal Chromaffin Cells by Similar Pathways
Authors: TerBush Daniel R. , Holz Ronald W.
Published: October 2006

Abstract: We compared the characteristics of secretion stimulated by EGTA‐buffered Ba2+‐ and Ca2+‐containing solutions in digitonin‐permeabilized bovine adrenal chromaffin cells. Half‐maximal secretion occurred at approximately 100 μM Ba2+ or 1 μM Ca2+. Ba2+‐stimulated release was not due to release of sequestered intracellular Ca2+ because at a constant free Ba2+ concentration, increasing unbound EGTA did not diminish the extent of release due to Ba2+. The maximal extents of Ba2+‐ and Ca2+‐dependent secretion in the absence of MgATP were identical. MgATP enhanced Ba2+‐induced secretion to a lesser extent than Ca2+‐induced secretion. Half‐maximal concentrations of Ba2+ and Ca2+, when added together to cells, yielded approximately additive amounts of secretion. Maximal concentrations of Ba2+ and Ca2+ when added together to cells for 2 or 15 min were not additive. Tetanus toxin inhibited Ba2+‐ and Ca2+‐dependent secretion to a similar extent. Ba2+, unlike Ca2+, did not activate polyphosphoinositide‐specific phospholipase C. These data indicate that (1) Ba2+ directly stimulates exocytosis, (2) Ba2+‐induced secretion is stimulated to a lesser extent than Ca2+‐dependent secretion by MgATP, (3) Ba2+ and Ca2+ use similar pathways to trigger exocytosis, and (4) exocytosis from permeabilized cells does not require activation of polyphosphoinositide‐specific phospholipase C.
Characterization of Histamine‐Induced Catecholamine Secretion from Bovine Adrenal Medullary Chromaffin Cells
Authors: Bunn Stephen J. , Boyd Teresa L.
Published: October 2006

Abstract: Histamine activation of H1 receptors stimulates 3H release from cultured bovine adrenal chromaffin cells preloaded with [3H]noradrenaline. The initial (1‐min) release induced by a high concentration of histamine was unaffected by the removal of extracellular Ca2+, whereas the more sustained response (10 min) was largely inhibited. In contrast, release induced by nicotine was dependent on extracellular Ca2+ at all times. The protein kinase inhibitor staurosporine inhibited both the initial and sustained (10‐min) phases of histamine‐induced release (IC50 in the region of 200 nM) but was ineffective against a direct depolarizing stimulus (56 mM K+). In contrast, the calmodulin antagonist trifluoperazine was equally effective against both stimuli. These data indicate that although a staurosporine‐sensitive event (perhaps involving protein kinase C) is essential for coupling histamine receptor activation to the release processes, it is not essential for exocytosis itself. A further distinction between histamine‐and depolarization‐induced release was demonstrated by the differential effect of the guanine nucleotide‐binding protein inhibitor pertussis toxin. Pretreatment with pertussis toxin (0.1 μg/ml for 16 h) enhanced depolarization‐induced release by ∼1.5‐fold. This pertussis toxin pretreatment was, however, approximately twofold as effective in potentiating histamine‐evoked release. Thus, the characteristics of the histaminergic response are distinct from those of a depolarizing stimulus, perhaps indicating the involvement of different mechanisms in the release process.
Opioid Signal Transduction in Intact and Fragmented SH‐SY5Y Neural Cells
Authors: Carter Bruce D. , Medzihradsky Fedor
Published: October 2006

Abstract: Parameters of ligand binding, stimulation of low‐Km GTPase, and inhibition of adenylate cyclase were determined in intact human neuroblastoma SH‐SY5Y cells and in their isolated membranes, both suspended in identical physiological buffer medium. In cells, the μ‐selective opioid agonist [3H]Tyr‐D‐Ala‐Gly(Me)Phe‐Gly‐ol ([3H]DAMGO) bound to two populations of sites with KD values of 3.9 and 160 nM, with <10% of the sites in the high‐affinity state. Both sites were also detected at 4°C and were displaced by various opioids, including quaternary naltrexone. The opioid antagonist [3H]naltrexone bound to a single population of sites, and in cells treated with pertussis toxin the biphasic displacement of [3H]naltrexone by DAMGO became monophasic with only low‐affinity binding present. The toxin specifically reduced high‐affinity agonist binding but had no effect on the binding of [3H]naltrexone. In isolated membranes, both agonist and antagonist bound to a single population of receptor sites with affinities similar to that of the high‐affinity binding component in cells. Addition of GTP to membranes reduced the Bmax for [3H]DAMGO by 87% and induced a linear ligand binding component; a low‐affinity binding site, however, could not be saturated. Compared with results obtained with membranes suspended in Tris buffer, agonist binding, including both receptor density and affinity, in the physiological medium was attenuated. The results suggest that high‐affinity opioid agonist binding represents the ligand‐receptor‐guanine nucleotide binding protein (G protein) complex present in cells at low density due to modulation by endogenous GTP. Opioid receptor coupling to adenylate cyclase in intact and fragmented cells occurred with similar efficiency: DAMGO inhibited adenylate cyclase with Ki, values of 11 nM in cells and 26 nM in lysates, with 30% maximal inhibition in both preparations. Receptor coupling to G protein in membranes occurred with similar parameters: DAMGO stimulated low‐Km GTPase with a Ks of 31 nM and an Smax of 48%. Both effector responses were blocked by naloxone and were strongly impaired by rigorous cell homogenization. These results indicate that opioid signal transduction in intact SH‐SY5Y cells and their appropriately isolated membranes functions with similar efficiencies involving a large reserve of uncoupled receptors.
Interleukin‐1‐β and Tumor Necrosis Factor‐α Increase Peripheral‐Type Benzodiazepine Binding Sites in Cultured Polygonal Astrocytes
Authors: Oh Young J. , Francis Jonathan W. , Markelonis George J. , Oh Tae H.
Published: October 2006

Abstract: Peripheral‐type benzodiazepine binding sites (PTBBS) are markedly increased in the injured CNS. Astrocytes appear to be the primary cell type which express increased PTBBS. Because certain cytokines within the injured CNS are potent mitogens for astrocytes, we examined the effects of two such cytokines, interleukin (IL)‐1β and tumor necrosis factor (TNF), on PTBBS in cultured astrocytes using [3H]Ro 5‐4864 as the specific ligand. Purified cultures of either polygonal or process‐bearing astrocytes were prepared from neonatal rat cerebral hemispheres. At a concentration of 1.8 nM, specific binding of the radioactive ligand to polygonal astrocytes reached equilibrium within 60 min and was half‐maximal by 5–10 min. By contrast, specific binding to process‐bearing astrocytes barely exceeded background levels. IL‐1 and TNF increased PTBBS within polygonal astrocytes in both dose‐ and time‐dependent manners. At 10–50 ng/ml, IL‐1β and TNF‐α elevated [3H]Ro 5–4864 binding in polygonal astrocyte cultures 65 and 87%, respectively, above the level in control cultures. However, no changes in PTBBS were seen within polygonal astrocytes after IL‐2 treatment. Scatchard analysis of saturation binding experiments suggested that the increase in PTBBS promoted by TNF was due to an increased number of binding sites present in polygonal astrocytes and not due to an increase in receptor affinity. Binding data suggested that PTBBS within cultures of process‐bearing astrocytes were virtually absent irrespective of the treatment. These in vitro data suggest that certain cytokines found in the injured brain may be involved in up‐regulating PTBBS within a particular subtype of astrocyte.
Amperozide, a Novel Antipsychotic Drug, Inhibits the Ability of d‐Amphetamine to Increase Dopamine Release In Vivo in Rat Striatum and Nucleus Accumbens
Authors: Ichikawa Junji , Meltzer Herbert Y.
Published: October 2006

Abstract: The in vivo effects of amperozide, a novel atypical antipsychotic drug, on the release of dopamine (DA) and the output of its metabolite, 3,4‐dihydroxyphenylacetic acid (DOPAC), were investigated in the striatum and the nucleus accumbens of awake, freely moving rats using microdialysis. Amperozide (2–10 mg/kg, s.c.) significantly increased extracellular levels of DA in both the striatum and nucleus accumbens in a dose‐dependent manner. It had a similar but lesser effect on extracellular DOPAC levels in both regions. d‐Amphetamine (2 mg/kg, s.c.) alone produced a very large (43‐fold) increase in DA release, together with a 70% decrease in DOPAC levels in both the striatum and the nucleus accumbens. Amperozide (1–5 mg/kg, s.c.) 30 min before d‐amphetamine (2 mg/kg) dose‐dependently attenuated d‐amphetamine‐induced DA release but had no effect on the ‐amphetamine‐induced decrease in extracellular DOPAC levels in both regions. The effect of amperozide on d‐amphetamine‐induced DA release in the nucleus accumbens may explain the inhibitory effect of amperozide on amphetamine‐induced locomotor activity. However, the failure of amperozide to block amphetamine‐induced stereotypy, despite marked inhibition of striatal DA release, suggests the need to reexamine the importance of striatal DA for amphetamine‐induced stereotypy.
Alterations of Benzodiazepine Receptor Binding in Tremor Rats with Absence‐Like Seizures
Authors: Shirasaka Yukiyoshi , Ito Masatoshi , Mikawa Haruki , Serikawa Tadao , Yamada Junzo
Published: October 2006

Abstract: Tremor rats begin to exhibit clinical or electrical absence‐like seizures after 6 weeks of age, and by 14 weeks of age, all have seizures. Central‐type benzodiazepine receptor binding was investigated in tremor rats and control rats, aged 4 weeks and 16 weeks. Significantly lower benzodiazepine receptor density and no differences in affinity were found in the hippocampus of the tremor rats in comparison with that of control rats at both ages. This abnormality is considered to be due to a tremor gene and may be the cause of absence‐like seizures in tremor rats. A significantly lower receptor density was found in the cerebellum at 4 weeks of age in the tremor rats than in the control rats. These changes may be related to tremorous movements in the tremor rats. Receptor density was significantly lower in the brainstems of tremor rats and control rats at 16 weeks of age than at 4 weeks of age, and the decrease was more marked in control rats. These facts may reflect a reduced decrease in the response to the dysfunction of γ‐aminobutyric acidergic neurons, or the function of the γ‐aminobutyric acid/benzodiaz‐epine receptor system may be secondarily increased to suppress seizures in 16‐week‐old tremor rats.