bluuebunny/crossref_metadata_2025 huggingface dataset:
Multiple Signalling Pathways Interact in the Regulation of Nerve Growth Factor Production in L929 Fibroblasts
Authors: D'Mello Santosh R. , Heinrich Gerhard
Published: October 2006

: Fibroblasts are one of several cell types producing nerve growth factor (NGF) in neuronal targets. In previous studies we found that NGF production is up‐regulated by 12‐O‐tetradecanoylphorbol 13‐acetate (TPA) and serum, down‐regulated by corticosterone, and unaffected by dibutyryl‐cyclic AMP (db‐cyclic AMP) in fibroblasts. As fibroblasts in vivo are likely to be exposed to regulatory effects by more than one of these agents at any given time, we examined the effects of combinations of them on NGF production using L929 fibroblasts as a model system. TPA and serum together stimulated NGF production 10‐fold more than either agent alone. Corticosterone reduced NGF mRNA and NGF production to < 10% of basal levels whether or not TPA or serum, or both, were present but not in the presence of the gluco‐corticoid antagonist RU486. Corticosterone did not increase the rate of NGF mRNA degradation. Forskolin and db‐cyclic AMP prevented NGF mRNA induction by TPA and serum without changing basal levels. TPA induced c‐fos and junB mRNAs transiently and preceding NGF mRNA induction but c‐jun mRNA remained undetectable. Forskolin enhanced the induction of both junB and c‐fos mRNA whereas corticosterone prolonged junB mRNA induction. Thus, TPA induction of NGF mRNA is modulated differentially by corticosterone and cyclic AMP. c‐fos and junB may play a role in the underlying mechanisms.
Stress‐and Endotoxin‐Induced Increases in Brain Tryptophan and Serotonin Metabolism Depend on Sympathetic Nervous System Activity
Authors: Dunn Adrian J. , Welch Jon
Published: October 2006

: Stressful treatments and immune challenges have been shown previously to elevate brain concentrations of tryptophan. The role of the autonomic nervous system in this neurochemical change was investigated using pharmacological treatments that inhibit autonomic effects. Pretreatment with the ganglionic blocker chlorisondamine did not alter the normal increases in catecholamine metabolites, but prevented the increase in brain tryptophan normally observed after footshock or restraint, except when the duration of the footshock period was extended to 60 min. The footshock‐and restraint‐related increases in 5‐hydroxyindoleacetic acid (5‐HIAA) were also prevented by chlorisondamine. The increases in brain tryptophan caused by intraperitoneal injection of endotoxin or interleukin‐1 (IL‐1) were also prevented by chlorisondamine pretreatment. The footshock‐induced increases in brain tryptophan and 5‐HIAA were attenuated by the β‐adrenergic antagonist propranolol but not by the α‐adrenergic antagonist phenoxybenzamine or the muscarinic cholinergic antagonist atropine. Thus the autonomic nervous system appears to be involved in the stress‐related changes in brain tryptophan, and this effect is due to the sympathetic rather than the parasympathetic limb of the system. Moreover, the main effect of the sympathetic nervous system is exerted on β‐as opposed to α‐adrenergic receptors. We conclude that activation of the sympathetic nervous system is responsible for the stress‐related increases in brain tryptophan, probably by enabling increased brain tryptophan uptake. Endotoxin and IL‐1 also elevate brain tryptophan, presumably by a similar mechanism. The increase in brain tryptophan appears to be necessary to sustain the increased serotonin catabolism to S‐HIAA that occurs in stressed animals, and which may reflect increased serotonin release.
Phosphorylation of Nuclear Proteins in Myelinating Oligodendrocytes and Its Control by Cyclic AMP
Authors: Sato‐Bigbee Carmen , Yu Robert K.
Published: October 2006

: Oligodendroglial nuclei isolated from rat brains at different stages of myelinogenesis (10,18, and 30 days of age) were incubated with (γ‐32P]ATP and extracted with 0.75 Mperchloric acid to yield a fraction of nonacidic chromatin proteins. The protein extracts were then analyzed by poly‐acrylamide gel electrophoresis. The phosphorylation pattern of these proteins was found to be different for different age groups. In 10‐day‐old rat Oligodendrocytes the most extensive phosphorylation occurred in low molecular mass species (<30 kDa), in contrast to fractions obtained from 18‐and 30‐day‐old rat Oligodendrocytes which showed a significantly higher labeling of the proteins with molecular masses >30 kDa. The phosphorylation of the latter species was greatly stimulated by the presence of cyclic AMP in the incubation media. The results suggest that the phosphorylation of specific nuclear proteins, which may play a regulatory role at different stages of oligodendroglial maturation and myelinogenesis, may be at least partially modulated by intracellular cyclic AMP.
Intracellular pH and Catecholamine Secretion from Bovine Adrenal Chromaffin Cells
Authors: Kao Lung‐Sen , Ho Mei‐Ying , Cragoe Edward J.
Published: October 2006

: To study the role of intracellular pH (pH1) in catecholamine secretion and the regulation of pHi in bovine chromaffin cells, the pH‐sensitive fluorescent indicator [2′,7′‐bis(carboxyethyl)‐5(6)‐carboxyfluorescein] was used to monitor the on‐line changes in pHi. The pHi of chromaffin cells at resting state is ⋍7.2. The pH1 was manipulated first by incubation of the cells with NH4+, and then the solution was replaced with a NH4+‐frce solution to induce acidification of the cytoplasm. The pH1 returned toward the basal pH value after acidification within 5–10 min in the presence of Na+or Li+, but the pH1 stayed acidic when Na+‐free buffers were used or in the presence of amiloride and its analogues. These results suggest that the pH recovery process after an acid load is due to the Na+/H+ exchange activity in the plasma membrane of the chromaffin cells. The Catecholamine secretion evoked by carbachol and Na+ removal was enhanced after the cytoplasm had been made more acidic. It appears that acidic pH favors the occurrence of exocytosis.
Lactate Utilization by Isolated Cells from Early Neonatal Rat Brain
Authors: Vicario Carlos , Arizmendi Carmen , Malloch Gavin , Clark John B. , Medina José M.
Published: October 2006

: The utilization of lactate, glucose, 3‐hydroxybutyrate, and glutamine has been studied in isolated brain cells from early newborn rats. Isolated brain cells actively utilized these substrates, showing saturation at concentrations near physiological levels during the perinatal period. The rate of lactate utilization was 2.5‐fold greater than that observed for glucose, 3‐hydroxybutyrate, or glutamine, suggesting that lactate is the main metabolic substrate for the brain immediately after birth. The apparent Km for glucose utilization suggested that this process is limited by the activity of hexokinase. However, lactate, 3‐hydroxybutyrate, and glutamine utilization seems to be limited by their transport through the plasma membrane. The presence of fatty acid‐free bovine serum albumin (BSA) in the incubation medium significantly increased the rate of lipogenesis from lactate or 3‐hydroxybutyrate, although this was balanced by the decrease in their rates of oxidation in the same circumstances. BSA did not affect the rate of glucose utilization. The effect of BSA was due not to the removal of free fatty acid, but possibly to the binding of long‐chain acyl‐CoA, resulting in the disinhibition of acetyl‐CoA carboxylase and citrate carrier.
Cyclic AMP Accumulation Induces a Rapid Desensitization of the Cyclic AMP‐Dependent Protein Kinase in Mouse Striatal Neurons
Authors: Chneiweiss Herve , Cordier Jocelyne , Glowinski Jacques
Published: October 2006

: Striatal neurons from the mouse brain embryo grown in primary culture express high levels of cyclic AMP (cAMP)‐dependent protein kinase (PK A) activity. To study the modulation of PKA in intact neurons, a rapid method based on Zn2+‐protein precipitation was developed. This strategy allowed analysis of the stimulation of PKA under conditions of intracellular cAMP concentration increases. Whereas increases up to 1 μM lead to an activation, large and sustained accumulations of cAMP result in a loss of the enzyme activity. With 8‐bromo‐cAMP (8‐Br‐cAMP) at 100 μM, the PKA refractoriness occurs within 2 min. It is rapidly reversible because incubation of treated neurons in fresh medium leads to a complete recovery of PKA activity within 30 min. The decrease in assayable PKA does not involve an activation of phosphatases because the histone dephosphorylation rate is not affected by 8‐Br‐cAMP treatment Finally, not only 8‐Br‐cAMP‐but also forskolin‐and vasoactive intestinal peptide‐induced increases in intracellular cAMP concentration can lead to the PKA desensitization. Altogether, these data unravel a new mechanism of PKA regulation.
Regulation of Cyclic AMP Levels by Calcium in Bovine Adrenal Medullary Cells
Authors: Keogh Rosemary , Marley Philip D.
Published: October 2006

: Both nicotine and histamine have been reported to increase cyclic AMP levels in chromaffin cells by Ca2+‐dependent mechanisms. The present study investigated whether Ca2+ was an adequate and sufficient signal for increasing cyclic AMP in cultured bovine adrenal medullary cells. Depolarization with 50 mM K+ caused a two‐to threefold increase in cellular cyclic AMP levels over 5 min, with no change in extracellular cyclic AMP. This response was abolished by omission of extracellular Ca2+ and by 100 μM methoxyverapamil, and was unaffected by 1 μM tetrodotoxin and by 1 mM isobutylmethylxanthine. Veratridine (40 μM) also increased cellular cyclic AMP levels by two‐to fourfold. This response was abolished by either methoxyverapamil or tetrodotoxin. The Ca2+ ionophore A23187 (10–50 μM) had little or no effect on cellular cyclic AMP levels. When the concentration of K+ used to depolarize the cells was reduced to 12–15 mM, the catecholamine release was similar to that induced by 50 μM A23187, and the cyclic AMP response was almost abolished. The results suggest that Ca2+ entry into chromaffin cells is a sufficient stimulus for increasing cellular cyclic AMP production. The possible involvement of a Ca2+/calmodulin‐dependent isozyme of adenylate cyclase is discussed.
Interactions Between the Argininyl Moieties of Neurotensin and the Catechol Protons of Dopamine
Authors: Schenk James O. , Morocco Martin T. , Ziemba Victoria A.
Published: October 2006

: The interaction between dopamine and neurotensin as well as other Arg‐containing peptides was studied to provide more chemical details of how dopamine binds to the neuropeptide neurotensin. The stoichiometry of 1:1, dopamine to neurotensin, was confirmed by additional electroanalytical and ultraviolet‐visible spectroscopic studies. By analyses of the 205‐to 340‐nm difference spectra of fixed concentrations of dopamine in the presence of increasing amounts of neurotensin, the dissociation constant of the interaction was found to be 5.9 × 10−8 mol/L. This finding confirmed (by a second physical method) the previously reported KD value obtained by electroanalytical techniques. The associations between dopamine and neurotensin as well as the neurotensin fragment Pro7‐Arg8‐Arg9‐Pro10 were found to be pH dependent when the dissociation constant was measured as a function of pH (in 150 mmol/L NaCl). The results of studies of the formal potential of dopamine in the presence of Arg and Arg‐containing peptides confirmed that catechol protons are directly involved in the association and that the chemical species of dopamine associated with neurotensin is a cate‐cholate form. The (pseudo)‐first‐order rate constant of dissociation of the complex at pH 7.6, measured by the chronoamperometric and rotating disk electroanalytical techniques, was found to be ⋍105 s−1, indicating that the rate of formation of the complex is under diffusion control. A hypothetical chemical structure of the neurotensin‐dopamine complex is suggested.
Hypothermia Prevents the Ischemia‐Induced Translocation and Inhibition of Protein Kinase C in the Rat Striatum
Authors: Cardell Monika , Boris‐Möller Fredrik , Wieloch Tadeusz
Published: October 2006

: The effect of hypothermia on the ischemia‐induced changes in the subcellular distribution of protein kinase C (PKC)(γ), ‐(βII), and ‐(α) and the activity of PKC was studied in striatal homogenates of rats subjected to 20 min of cerebral ischemia. The effect of post‐ischemic cooling was also studied. During normothermic ischemia, PKC(γ) and ‐(βII) increased 3.9‐and 2.9‐fold, respectively, in the particulate fraction, signifying a translocation of PKC to cell membranes. The levels of PKC(α) did not change significantly. PKC activity decreased during ischemia by 52% and 47% (p < 0.05) in the paniculate and cytosolic fractions, respectively, and remained inhibited for the 1 h recovery period. In hypothermic animals, there was no evidence of translocation, and the inhibition of PKC activity was completely abolished. Hypothermia induced in the recovery phase, however, did not affect PKC distribution or activity. The protective effect of intraischemic hypothermia may in part be due to the prevention of the ischemia‐induced translocation and subsequent downregulation of PKC, possibly through a temperature‐dependent modification of the cell membranes.
Regulation of 109Cd2+ Uptake into Isolated Neurohypophysial Peptidergic Nerve Terminals
Authors: Toescu Emil C.
Published: October 2006

Cadmium (109Cd2+) uptake was studied in a preparation of isolated neurohypophysial nerve terminals. By use of a filter separation method, together with a permeabilizing agent (Triton X‐100), two cellular Cd2+ pools have been distinguished. The uptake into the intraterminal pool was governed mainly by a process that displayed saturable kinetics, with a Vmax of 0.15 nmol of Cd2+/mg of protein /min and a Km of 0.18 mM, consistent with a transport system. The superficially bound Cd2+ pool (Triton insensitive), which represented 30–50% of the total Cd2+ bound to the cellular system, was very sensitive to the ionic composition of the incubation medium. Reducing the extracellular Ca2+ or Na+ concentration caused a significant increase in the size of the Triton‐insensitive Cd2+ pool. Whereas Na+ did not affect Cd2+ uptake, Ca2+ induced a small, but significant, increase of Cd2+ uptake into the terminals. It is concluded that there is a significant intraterminal uptake of Cd2+, which could explain several physiological effects of this ion.
Suppression of In Vivo Neostriatal Acetylcholine Release by Vesamicol: Evidence for a Functional Role of Vesamicol Receptors in Brain
Authors: Marien Marc R. , Richard Jean W. , Allaire Catherine , Altar C. Anthony
Published: October 2006

Experiments examined the effects of peripheral and central administration of the vesicular acetylcholine transport blocker vesamicol (AH5183) on the content, synthesis, and release of acetylcholine in the rat brain in vivo. In time course studies, a single intraperitoneal dose of dl‐vesamicol (5 mg/kg) rapidly and reversibly (within 2 h) doubled the content of acetylcholine in the striatum and hippocampus, without affecting choline levels or the rate of transmitter synthesis. In microdialysis experiments, the same peripheral dose of drug produced a reversible 55% reduction in endogenous striatal acetylcholine release. A similar inhibitory effect was produced by direct intrastriatal perfusion with vesamicol. Moreover, this effect of vesamicol was (a) concentration‐dependent and saturable (EC50= 68 nM), (b) rapidly reversible, (c) stereospecific for the l‐isomer, and (d) poorly mimicked by a vesamicol analog with lower plasma membrane permeability. This profile of effects is consistent with an interaction with a specific vesamicol receptor as defined by previous in vitro binding studies. These results support a functional role for vesamicol receptors in modulating central cholinergic transmission in vivo.
1‐Methyl‐ 1,2,3,4‐Tetrahydroisoquinoline, Decreasing in 1‐Methyl‐4‐Phenyl‐1,2,3,6‐Tetrahydropyridine‐Treated Mouse, Prevents Parkinsonism‐Like Behavior Abnormalities
Authors: Tasaki Yoshikazu , Makino Yukiko , Ohta Shigeru , Hirobe Masaaki
Published: October 2006

In l‐methyl‐4‐phenyl‐l,2,3,6‐tetrahydropyridine (MPTP)‐treated mouse brain, there was no significant increase or decrease in the content of an endogenous amine, 1,2,3,4‐tctrahydroisoquinoline (TIQ), which is well noted for inducing parkinsonism, whereas another endogenous amine, 1‐methyl‐1,2,3,4‐tetrahydroisoquinoline (1‐MeTIQ), was markedly reduced. This result agrees with the finding in human idiopathic parkinsonianism, confirmed by our previous research. In addition, pretreatment with 1‐MeTIQ completely prevented MPTP‐ or TIQ‐inducing bradykinesia, a symptom of parldnsonism. This study confirmed that 1‐MeTIQ plays an important role in preventing the pathogenesis of parldnsonism and is a possible leading compound of anti‐parkinsonism agents.
Two Possibly Distinct Prostaglandin E1 Receptors in N1E‐115 Clone: One Mediating Inositol Trisphosphate Formation, Cyclic GMP Formation, and Intracellular Calcium Mobilization and the Other Mediating Cyclic AMP Formation
Authors: Kanba Shigenobu , Sasakawa Nobuyuki , Nakaki Toshio , Kanba Kiyoko‐Shimizu , Yagi Gohei , Kato Ryuichi , Richelson Elliott
Published: October 2006

Prostaglandin E1 (PGE1)‐mediated transmembrane signal control systems were investigated in intact murine neuroblastoma cells (clone N1E‐115). PGE1 increased intra‐cellular levels of total inositol phosphates (IP), cyclic GMP, cyclic AMP, and calcium ([Ca2+]i). PGE1 transiently increased inositol 1,4,5‐trisphosphate formation, peaking at 20 s. There was more than a 10‐fold difference between the ED50 for PGE1 at cyclic AMP formation (70 nM) and its ED50 values at IP accumulation (1 μM), cyclic GMP formation (2 μM), and[Ca2+]i increase (5 μM). PGE1‐mediated IP accumulation, cyclic GMP formation, and [Ca2+]i increase depended on both the concentration of PGE1 and extracellular calcium ions. PGE1 had more potent intrinsic activity in cyclic AMP formation, IP accumulation, and cyclic GMP formation than did PGE2, PGF2α, or PGD2. A protein kinase C activator, 4β‐phorbol 12β‐myristate 13α‐acetate, had opposite effects on PGE1‐mediated IP release and cyclic GMP formation (inhibitory) and cyclic AMP formation (stimulatory). These data suggest that there may be subtypes of the PGE1 receptor in this clone: a high‐affinity receptor mediating cyclic AMP formation, and a low‐affinity receptor mediating IP accumulation, cyclic GMP formation, and intracellular calcium mobilization.
Synergistic Regulation of Cytosolic Ca2+ Concentration in Mouse Astrocytes by NK1 Tachykinin and Adenosine Agonists
Authors: Delumeau Jean C. , Petitet François , Cordier Jocelyne , Glowinski Jacques , Prémont Joël
Published: October 2006

The effects on cytosolic Ca2+ concentration of 2‐chloroadenosine and [L‐Pro9]‐substance P, a selective agonist of NK1 receptors, were investigated on astrocytes from embryonic mice in primary culture. Cells responded to [L‐Pro9]‐ substance P with a transitory increase in cytosolic Ca2+ which was of shorter duration when external Ca2+ was removed. A transient response to 2‐chloroadenosine alone occurred. When simultaneously applied, [L‐Pro9]‐substance P and 2‐chloroadenosine evoked a prolonged elevation of cytosolic Ca2+ (up to 30 min). This phenomenon was dependent on the presence of extracellular Ca2+, but insensitive to dihy‐dropyridines, La3+, and Co2+, excluding the implication of voltage‐operated Ca2+ channels. Arachidonic acid also induced a sustained elevation of cytosolic Ca2+, but did not increase further the response evoked by [L‐Pro9]‐substance P and 2‐chloroadenosine. The activation of protein kinase C by a diacylglycerol analogue mimicked the effect of [L‐Pro9]‐substance P in potentiating the 2‐chloroadenosine‐evoked response. Like 2‐chloroadenosine, pinacidil, which hyper‐polarizes the cells by opening K+ channels, prolonged the elevation of cytosolic Ca2+ concentration induced by [L‐Pro9]‐substance P. Conversely, depolarization with 50 mM KC1 canceled the effects of either pinacidil or 2‐chloroadenosine applied with [L‐Pro9]‐substance P. Pertussis toxin pretreatment suppressed all the effects induced by 2‐chloroadenosine.
Molecular Species of Diacylglycerols and Phosphoglycerides and the Postmortem Changes in the Molecular Species of Diacylglycerols in Rat Brains
Authors: Lee Chunghee , Hajra Amiya K.
Published: October 2006

Abstract: The molecular species of 1,2‐diacyl‐sn‐glycerol (DAG), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidylinositol 4‐phosphate (PIP), and phosphatidylinositol 4,5‐bisphosphate (PIP2) from brains of adult rats (weighing 150 g) were determined. The DAG, isolated from brain lipid extracts by TLC, was benzoylated, and the molecular species of the purified benzoylated derivatives were separated from each other by reverse‐phase HPLC. The total amount and the concentration of each species were quantified by using 1,2‐distearoyl‐sn‐glycerol (18:0–18:0) as an internal standard. About 30 different molecular species containing different fatty acids at the sn‐1 and sn‐2 positions of DAG were identified in rat brains (1 min postmortem), and the predominant ones were 18:0–20:4 (35%), 16:0–18:1 (15%), 16:0–16:0 (9%), and 16:0–20:4 (8%). The molecular species of PC, PE, PS, and PI were determined by hydrolyzing the lipids with phospholipase C to DAG, which was then benzoylated and subjected to reverse‐phase HPLC, PIP and PIP2 were first dephosphorylated to PI with alkaline phosphatase before hydrolysis by phospholipase C. The molecular species composition of phosphoinositides showed predominantly the 18:0–20:4 species (50% in PI and ∼65% in PIP and PIP2). PS contained mainly the 18:0–22:6 (42%) and 18:0–18:1 (24%) species. PE was mainly composed of the 18:0–20:4 (22%), 18:0–22:6 (18%), 16:0–18:1 (15%), and 18:0–18:1 (15%) species. In PC the main molecular species were 16:0–18:1 (36%), 16:0–16:0 (19%), and 18:0–18:1 (14%). Studies on postmortem brains (30 s to 30 min) showed a rapid increase in the total amount (from 40–50 nmol/g in 0 min to 210–290 nmol/g in 30 min) and in all the molecular species of DAG. Comparatively larger increases (seven‐ to 10‐fold) were found for the 18:0–20:4 and 16:0–20:4 species. Comparison of DAG species with the molecular species of different glycerolipids indicated that the rapid postmortem increase in content of DAG was mainly due to the breakdown of phosphoinositides. However, a slow but continuous breakdown of PC to DAG was also observed.
Differential Subcellular Distribution of p36 (the Heavy Chain of Calpactin I) and Other Annexins in the Adrenal Medulla
Authors: Drust Debra S. , Creutz Carl E.
Published: October 2006

Abstract: The annexins are a group of highly related Ca2+‐dependent membrane‐binding proteins that are present in a wide variety of cells and tissues. We have examined the subcellular distribution of five members of the annexin family in the adrenal medulla. Bovine adrenal medullary tissue was homogenized in buffers containing EGTA and fractionated on sucrose gradients. p36 (the large subunit of calpactin I) was found to be predominantly membrane associated, with ∼20% present in fractions enriched in chromaffin granules. In contrast, lipocortin I was localized primarily to the cytosol, with only a small proportion found in plasma membrane‐containing fractions. Like lipocortin I, endonexin I was found to be present almost entirely in the soluble fractions. The 67‐kDa calelectrin was localized primarily to the plasma membrane fractions, with a small amount present in the chromaffin granule and cytoplasmic fractions. Synexin was present in both membranous and cytoplasmic fractions. p36 appeared to be a peripherally associated granule membrane protein in that it was dissociated from the membrane by addition of base and it partitioned with the aqueous phase when granule membranes were treated with Triton X‐114. Antiserum against p10 (the small subunit of calpactin I) reacted with a protein of 19 kDa that is specifically localized in chromaffin granule membrane fractions. The differences in subcellular distributions of the annexins suggest that these proteins have distinct cellular functions. The finding that p36 is associated with chromaffin granule and plasma membrane fractions provides further support for a possible role of calpactin in exocytosis.
Characterization of Prostaglandin E2‐Induced Ca2+ Mobilization in Single Bovine Adrenal Chromaffin Cells by Digital Image Microscopy
Authors: Ito Seiji , Mochizuki‐Oda Noriko , Hori Kunio , Ozaki Kazuho , Miyakawa Atsuo , Negishi Manabu
Published: October 2006

Abstract: We recently reported that prostaglandin E2 (PGE2) stimulates phosphoinositide metabolism accompanied by an increase in intracellular free Ca2+ concentration ([Ca2+]i) in cultured bovine adrenal chromaffin cells. In the present study, temporal and spatial changes in [Ca2+]i induced by PGE2 in fura‐2‐loaded individual cells were investigated by digital image microscopy and were compared with those induced by nicotine and histamine. Image analysis of single cells revealed that responses to PGE2 showed asynchrony with the onset of [Ca2+]i changes. After a lag time of 10–30 s, PGE2‐induced [Ca2+]i changes took a similar prolonged time course in almost all cells: a rapid rise followed by a slower decline to the basal level over 5 min. Few cells exhibited oscillations in [Ca2+]i. In contrast, nicotine and histamine induced rapid and transient [Ca2+]i changes, and these [Ca2+]i changes were characteristic of each stimulant. Whereas pretreatment of the cells with pertussis toxin (100 ng/ml, 6 h) did not block the response to any of these stimulants, treatment with 12‐O‐tetra‐decanoylphorbol 13‐acetate (100 nM, 10 min) completely abolished [Ca2+]i changes elicited by PGE2 and histamine. In a Ca2+‐free medium containing 3 mM EGTA, or in medium to which La3+ was added, the [Ca2+]i response to nicotine disappeared, but that to histamine was not affected significantly. Under the same conditions, the percentage of the cells that responded to PGE2 was reduced to 37% and the prolonged [Ca2+]i changes induced by PGE2 became transient in responding cells, suggesting that the maintained [Ca2+]i increase seen in normal medium is the result of a PGE2‐stimulated entry of extracellular Ca2+. Whereas the organic Ca2+‐channel blocker nicardipine inhibited [Ca2+]i changes by all stimulants at 10 μM, these [Ca2+]i changes were not affected by any of the organic Ca2+‐channel blockers, i.e., verapamil, diltiazem, nifedipine, and nicardipine, at 1 μM, a concentration high enough to inhibit voltage‐sensitive Ca2+ channels. These results demonstrate that PGE2 may promote Ca2+ entry with concomitant release of Ca2+ from intracellular stores and that the mechanism(s) triggered by PGE2 is apparently different from that by histamine or nicotine.
Differential Interaction of Phencyclidine‐Like Drugs with the Dopamine Uptake Complex In Vivo
Authors: Maurice Tangui , Vignon Jacques , Kamenka Jean‐Marc , Chicheportiche Robert
Published: October 2006

Abstract: The phencyclidine (PCP) derivative, [3H]N‐[1‐(2‐benzo[b]thiophenyl)cyclohexyl]piperidine ([3H]BTCP), was used to label in vivo the dopamine uptake complex in mouse brain. The striatum accumulated the highest level of total and specific binding. Drugs which bind to the dopamine uptake site inhibited [3H]BTCP binding on an order similar to their in vitro affinities for the high‐affinity [3H]BTCP site. Drugs which label selectively other monoamine uptake complexes, PCP, or σ recognition sites were ineffective at doses up to 40 mg/kg. PCP bound to and dissociated from the dopamine uptake complex very rapidly. N‐[1‐(2‐Thienyl)cyclohexyl]piperidine (TCP) and (+)‐5‐methyl‐10,11‐dihydro‐5H‐dibenzo[a,d]cyclohepten‐5,10‐imine maleate (MK‐801) had no effect at any time or at any dose. These results imply that the pharmacological effects of PCP are due to its simultaneous interaction with the dopamine uptake complex and the PCP receptor. Conversely, TCP and MK‐801, which have the same behavioral properties as PCP, exert their action only through the interaction with the PCP receptor.
Myelin Basic Protein Gene and the Function of Antisense RNA in Its Repression in Myelin‐Deficient Mutant Mouse
Authors: Okano Hideyuki , Aruga Jun , Nakagawa Toshiyuki , Shiota Chiyo , Mikoshiba Katsuhiko
Published: October 2006

Abstract: The myelin‐deficient (mld) mouse is an autosomal recessive mutant characterized by hypomyelination of the CNS due to reduced expression of the myelin basic protein (MBP) gene. In the mld mutant, the MBP gene is duplicated in tandem. One gene is intact, but a large portion is inverted upstream of the other copy, and its transcription yields the antisense RNA. This antisense RNA was shown to be localized in the nucleus and to form an RNA:RNA duplex with sense RNA. These findings suggested that inhibition of transport from the nucleus or selective degradation of the duplex is responsible for the reduced expression of the MBP gene in the mld mutant. The mechanism of gene rearrangement at the MBP locus was also characterized. Cosmid clones encompassing whole MBP gene loci from control and mld genomic DNA libraries were isolated. The recombination points indicated that the duplication and inversion observed in mld occurred due to nonhomologous recombination.
Nicotine Indirectly Inhibits [3H]Dopamine Uptake at Concentrations That Do Not Directly Promote [3H]Dopamine Release in Rat Striatum
Authors: Izenwasser Sari , Jacocks Henry M. , Rosenberger John G. , Cox Brian M.
Published: October 2006

Abstract: The effects of both (–)‐ and (+)‐nicotine isomers were examined on in vitro uptake and release of [3H]dopamine in rat striatum. Both isomers inhibited uptake of [3H]dopamine in chopped tissue at concentrations well below those necessary for promoting release of preloaded [3H]dopamine. (–)‐Nicotine was more potent than (+)‐nicotine both at inhibiting uptake and at promoting release. Unlike other dopamine uptake inhibitors, however, nicotine inhibited only 50% of the total uptake. In the presence of 1 nM nicotine, the residual [3H]dopamine uptake was less sensitive to inhibition by cocaine than uptake in the absence of nicotine. Nicotine did not compete against the binding of [3H]GBR 12935, a selective dopamine uptake inhibitor. The nicotinic receptor agonists carbachol and 1,1‐dimethyl‐4‐phenylpiperazinium iodide also inhibited uptake, whereas the nicotinic antagonists chlorisondamine and mecamylamine blocked nicotine's effect. Thus, the effect of nicotine on dopamine uptake appears to be mediated by a receptor similar to the nicotinic acetylcholine receptor. These receptors do not seem to be on the terminals that are accumulating dopamine, however, since tetrodotoxin prevented the effect of nicotine on [3H]dopamine uptake and nicotine had no effect on uptake in a synaptosomal preparation.
ω‐Conotoxin Inhibits the Acute Activation of Tyrosine Hydroxylase and the Stimulation of Norepinephrine Release by Potassium Depolarization of Sympathetic Nerve Endings
Authors: Rittenhouse Ann R. , Zigmond Richard E.
Published: October 2006

Abstract: Increased Ca2+ influx serves as a signal that initiates multiple biochemical and physiological events in neurons following depolarization. The most widely studied of these phenomena is the release of neurotransmitters. In sympathetic neurons, depolarization also increases the rate of synthesis of the transmitter norepinephrine (NE), via an activation of the enzyme tyrosine hydroxylase (TH), and this effect also seems to involve Ca2+ entry. We have examined whether the mechanism of Ca2+ entry relevant to TH activation is via voltage‐sensitive Ca2+ channels and, if so, whether the type of Ca2+ channel involved is the same as that involved in the stimulation of NE release. We have investigated the isolated rat iris, allowing us to examine transmitter biosynthesis and release in sympathetic nerve terminals in the absence of sympathetic cell bodies and dendrites. Potassium depolarization produced a three‐ to fivefold increase in TH activity and an ∼ 100‐fold increase in NE release. Both effects were dependent on Ca2+ being present in the extracellular medium, and both were inhibited by ω‐conotoxin (1μM), which inhibits N‐type voltage‐sensitive Ca2+ channels. In contrast, the di‐hydropyridine nimodipine (1–3 μM), which blocks L‐type Ca2+ channels, had no effect on either measure. These data support the hypothesis that increases in NE biosynthesis and release in sympathetic nerve terminals during periods of depolarization are both initiated by an influx of Ca2+ through voltage‐sensitive Ca2+ channels and that a similar type of Ca2+ channel is involved in both processes.
Protein Kinase C and Phospholipase C Mediate α‐ and β‐Adrenoceptor Intercommunication in Rat Hypothalamic Slices
Authors: Petitti Nicolas , Etgen Anne M.
Published: October 2006

Abstract: These experiments examined the mechanism by which phenylephrine enhances β‐adrenoceptor‐stimulated cyclic AMP formation in rat hypothalamic and preoptic area slices. To this end we manipulated phospholipase C, phospholipase A2, and protein kinase C activity in slices and assessed the effects of these manipulations on phenylephrine augmentation of isoproterenol‐stimulated cyclic AMP generation. Since previous work indicated that estrogen enhances the α1‐component of cyclic AMP formation, we examined slices from both gonadectomized and estrogen‐treated animals. The α1‐antagonist prazosin eliminated phenylephrine augmentation of the β‐response, suggesting that α1‐adrenergic receptors mediate the potentiation of cyclic AMP formation. Inhibition of protein kinase C by H7 attenuated the α1‐augmentation of β‐stimulated cyclic AMP formation. Staurosporine, a more potent protein kinase C inhibitor, completely abolished the α1‐augmenting response. In addition, phenylephrine potentiation of the isoproterenol response was not observed if protein kinase C was first stimulated directly with a synthetic diacylglycerol (1‐oleoyl‐2‐acetyl‐sn‐glycerol) or phorbol ester (phorbol 12, 13‐dibutyrate). Neomycin, an inhibitor of phospholipase C, decreased α1‐receptor enhancement of β‐stimulated cyclic AMP formation, whereas quinacrine, an inhibitor of phospholipase A2, did not. The data suggest that the postreceptor mechanism involved in α1‐adrenergic receptor potentiation of cyclic AMP generation in hypothalamic and preoptic area slices includes activation of phospholipase C and protein kinase C.
Comparison of Solubilised Kainate and α‐Amino‐3‐Hydroxy‐5‐Methylisoxazolepropionate Binding Sites in Chick Cerebellum
Authors: Henley Jeremy M. , Barnard Eric A.
Published: October 2006

Abstract: [3H]Kainate bound to chick cerebellar membranes with a KD of 0.6 μM and with an exceptionally high Bmax of 165 pmol/mg of protein. In octylglucoside‐solubilised extracts, the affinity of [3H]kainate was reduced (KD= 2.7 μM), but the Bmax was relatively unchanged (130 pmol/mg of protein). The rank potency of competitive ligands was domoate > kainate > 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX) > glutamate. Binding sites for α‐[3H]amino‐3‐hydroxy‐5‐methylisoxazolepropionate ([3H]AMPA) were much less abundant, with KD and Bmax values in membranes of 86 nM and I pmol/mg of protein, respectively. The affinity of [3H]AMPA binding was also reduced on solubilisation (KD= 465 nM), but there was an increase in the Bmax (1.7 pmol/mg of protein). Quisqualate and CNQX were the most effective displacers of [3H]AMPA binding, but kainate was also a relatively potent inhibitor. However, in contrast to the displacement profile for [3H]kainate, domoate was markedly less potent than kainate at displacing [3H]AMPA. These results suggest that [3H]AMPA binds to a small subset of the kainate sites that, unlike the majority of the [3H]kainate binding protein, which has been reported to be located in the Bergmann glia, may represent neuronal unitary non‐N‐methyl‐D‐aspartate receptors.
Core Protein of Chondroitin Sulfate Proteoglycan Promotes Neurite Outgrowth from Cultured Neocortical Neurons
Authors: Iijima Noboru , Oohira Atsuhiko , Mori Toshio , Kitabatake Katsuaki , Kohsaka Shinichi
Published: October 2006

Abstract: Chondroitin sulfate proteoglycan (CS‐PG) was purified from rat brain and examined for its effect on neurite outgrowth in primary cultures of embryonic rat neocortical neurons. Neurite outgrowth was increased in culture wells coated with CS‐PG. The core protein and glycosaminoglycan (GAG) prepared from the CS‐PG were also examined for neurite‐promoting activity. The activity was observed in culture wells coated with the core protein but not with GAG. These results suggest that CS‐PG stimulates neurite outgrowth from the cultured neurons via its core protein.
Expression and Agonist‐Induced Down‐Regulation of mRNAs of m2‐ and m3‐Muscarinic Acetylcholine Receptors in Cultured Cerebellar Granule Cells
Authors: Fukamauchi Fumihiko , Hough Christopher , Chuang De‐Maw
Published: October 2006

Abstract: The regulation and expression of muscarinic acetylcholine receptor (mAChR) mRNA was studied in cultured cerebellar granule cells using Northern blot hybridization. mRNA species for m2‐ and m3‐mAChRs but not ml‐ and m4‐mAChRs were detected in these cells. The expression of mRNAs of both m2‐ and m3‐mAChRs reached a maximum on the tenth day in culture but their expression patterns differed. Treatment of cerebellar granule cells after 8 days in culture with 100 μM carbachol led to differential down‐regulation of the mRNA species of both mAChR subtypes present. Muscarinic receptor antagonists, atropine (1 μM) and pirenzepine (10 μM), prevented carbachol‐induced m3‐mAChR mRNA down‐regulation observed at 8 h. However, exposure to either atropine or pirenzepine alone for 8 h led to a significant up‐regulation of m3‐mAChR mRNA. Thus, the mRNA species for both m2‐ and m3‐mAChR subtypes are differentially expressed in culture and down‐regulated by agonist stimulation. The loss of these mRNA species may play a role in the down‐regulation of mAChR binding sites that occurs after desensitization.
The Posttranslational Processing of β‐Endorphin in Human Hypothalamus
Authors: Millington William R. , Smith Debra L.
Published: October 2006

β‐Endorphin is posttranslationally processed to six derivatives, which, although structurally similar, produce distinctly different biological effects. β‐Endorphin 1–31 is a potent opioid receptor agonist, but β‐endorphin 1–27 exhibits antagonist properties, and β‐endorphin 1–26 and the α‐N‐ acetyl derivatives of all three peptides lack opioid receptor activity. In the present study, we identified the β‐endorphin peptides synthesized in human hypothalamus using cation exchange HPLC. First, we tested whether postmortem changes occur by storing rat hypothalami at 4°C. This demonstrated that relative amounts of the six β‐endorphin forms did not change for up to 24 h, although total β‐endorphin immunoreactivity significantly declined after 6 h. HPLC analysis of human hypothalami revealed that β‐endorphin 1–31 was the principal form, constituting 58.4 ± 5.4% of total immunoreactivity. Substantial amounts of β‐endorphin 1–27 (13.4 ± 1.2%) and β‐endorphin 1–26 (13.1 ± 1.6%) were also present, but α‐N‐acetylated forms were quantitatively minor, each comprising ∼5% of total β‐endorphin. A similar processing pattern occurred in preoptic and supra‐ chiasmatic areas of the hypothalamus. These results show that, despite differences in primary sequence, β‐endorphin is processed similarly in both rat and human hypothalamus. Opiate‐active β‐endorphin 1–31 is the principal form in both species.
Polyamines Modulate the Binding of [3H]MK‐801 to the Solubilized N‐Methyl‐D‐Aspartate Receptor
Authors: Romano Carmelo , Williams Keith , Molinoff Perry B.
Published: October 2006

Spermine and spermidine enhance the binding of [3H](+)‐5‐methyl‐10,11‐dihydro‐5H‐dibenzo[a, d]cyclohepten‐5,10‐imine ([3H]MK‐801) to N‐methyl‐D‐aspartate (NMDA) receptors in membranes prepared from rat brain. These polyamines also enhance binding of [3H]MK‐801 to NMDA receptors that have been solubilized with deoxycholate. Other polyamines selectively antagonize this effect, a finding indicating that the polyamine recognition site retains pharmacological and structural specificity after solubilization. In the presence of spermidine, an increase in the affinity of the solubilized NMDA receptor for [3H]MK‐801 is observed. However, the rates of both association and dissociation of [3H]MK‐801 binding to solubilized NMDA receptors are accelerated when assays are carried out in the presence of spermidine. When kinetic data are transformed, pseudo‐first‐order association and first‐order dissociation plots are nonlinear in the presence of spermidine, an observation indicating a complex binding mechanism. Effects of spermidine on solubilized NMDA receptors are similar to effects previously described in studies of membrane‐bound receptors. The data indicate that polyamines interact with a specific recognition site that remains associated with other components of the NMDA receptor complex after detergent solubilization.
Glucocorticoids Regulate the Synthesis of Glial Fibrillary Acidic Protein in Intact and Adrenalectomized Rats but Do Not Affect Its Expression Following Brain Injury
Authors: O'Callaghan James P. , Brinton Roberta E. , McEwen Bruce S.
Published: October 2006

Short (5 days)‐to long‐term (4 months) corticosterone (CORT) administration by injection, pellet implantation, or in the drinking water decreased glial fibrillary acidic protein (GFAP) by 20–40% in hippocampus and cortex of intact rats. In contrast to CORT, adrenalectomy (ADX) caused elevations (50–125%) in hippocampus and cortex GFAP within 12 days of surgery that persisted for at least 4 months. CORT replacement of ADX rats decreased GFAP amount in hippocampus and cortex. The effects of long‐term CORT and ADX on GFAP in hippocampus and cortex were also seen in striatum, midbrain, and cerebellum, findings suggestive of brain‐wide adrenal steroid regulation of this astrocyte protein. The changes in GFAP amount due to CORT and ADX were paralleled by changes in GFAP mRNA, indicating a possible transcriptional or at least genomic effect of adrenal steroids. Glucocorticoid regulation of GFAP was relatively specific; it could not be generalized to other astrocyte proteins or other major structural proteins of neurons. The negative regulation of GFAP and GFAP mRNA by adrenal steroids suggested that increases in GFAP that result from brain injury may be attenuated by glucocorticoids. However, chronic CORT treatment of intact rats did not reverse or reduce the large increases in GFAP caused by trauma‐or toxicant‐induced brain damage. Thus, glucocorticoids and injury appear to regulate the expression of GFAP through different mechanisms. In contrast to the lack of effects of CORT on brain damage‐induced increases in GFAP, CORT treatment begun in 2‐week ADX rats, after an increase in GFAP had time to occur, did reverse the ADX‐induced increase in GFAP. These results suggest that the increase in GFAP resulting from ADX is not mediated through an injury‐linked mechanism.
Pre‐and Postnatal Developmental Changes of Adrenoceptor Subtypes in Rat Brain
Authors: Erdtsieck‐Ernste B. H. W. , Feenstra M. G. P. , Boer G. J.
Published: October 2006

β‐Adrenergic receptor subtypes, β1 and β2, were studied during pre‐and postnatal development in the rat brain. [125I]Iodocyanopindolol (6–300 pmol/L) binding assays in the presence of 5‐hydroxytryptamine (0.6–6 μmol/L) were used to measure exclusively β‐adrenergic receptors. In forebrain tissue, saturable and stereoselective binding was detected on gestational day 13. The amount of β‐adrenergic binding increased until postnatal day 23, when adult values were reached. The dissociation constants of [125I]iodocyanopindolol binding remained the same throughout development, as did the affinity of several β‐adrenergic and non‐β‐adrenergic compounds. The proportion of the β2‐adrenergic receptors was determined using the β1‐selective antagonist ICI‐89406 (7–150 nmol/L) and was found to change from 65% in prenatal forebrain tissue to 28% in adulthood. In cerebellum/medulla pons tissue, however, the proportion of β2‐receptor binding (80%) remained unchanged during the whole developmental period.
Neuropeptide Y: An Endogenous Inhibitor of Norepinephrine‐Stimulated Melatonin Secretion in the Rat Pineal Gland
Authors: Olcese James
Published: October 2006

The biosynthesis of the hormone melatonin (MEL) by the mammalian pineal gland has been thought to be regulated strictly by stimulatory factors, most predominantly norepinephrine (NE), released from the sympathetic nerve fibers which heavily innervate the gland. Evidence from many investigators suggests that sympathetic fibers may colocalize other neuroactive factors in addition to NE. One of these factors is neuropeptide Y (NPY), which has been found in the nerve fibers of the pineal gland. The present study sought to explore potential interactions between NE and NPY in the regulation of pineal MEL secretion. Specific, saturable, and reversible binding of 125I‐NPY to intact cultured pine‐ alocytes was measured with an affinity constant of 1 nM and an NPY binding site density of 0.04 pmol/mg of protein. In addition, cell culture studies revealed that NPY represents a potent (IC50 of 0.4 nM) endogenous inhibitor of NE‐stimulated MEL secretion. However, this inhibition is accompanied by only a modest reduction (35%) of cyclic AMP accumulation. These findings reinforce the view that the mammalian pineal gland, which appears to integrate both inhibitory as well as stimulatory signals, is an important model of autonomic function, particularly in the context of biological rhythmicity.
K‐252b Is a Selective and Nontoxic Inhibitor of Nerve Growth Factor Action on Cultured Brain Neurons
Authors: Knüsel Beat , Hefti Franz
Published: October 2006

K‐252b is a kinase inhibitor structurally related to K‐252a, which is known to abolish selectively the effects of nerve growth factor (NGF) on PC12 cells and PNS neurons. We tested whether K‐252b, K‐252a, and staurosporine, another related compound, are effective and selective inhibitors of NGF actions on CNS neurons. All three compounds, at appropriate concentrations, completely and selectively prevented the NGF‐mediated activity increase of the cholinergic marker enzyme choline acetyltransferase in cultures of rat basal forebrain cells. The stimulatory effects of basic fibroblast growth factor and insulin on choline acetyltransferase in these cultures and on dopamine uptake in cultures of dissociated ventral mesencephalon were not affected. No signs of toxicity were observed in cultures treated with K‐252b. In contrast, K‐252a and staurosporine, at concentrations required to block the NGF actions on cholinergic cells, were cytotoxic and produced cell loss. In addition, K‐252a, at higher concentrations and in the absence of growth factors, increased cell numbers. Our study suggests that K‐252b is a selective and nontoxic inhibitor of NGF actions in the brain and may become a useful tool to study these actions in vivo.
Effects of Intranigral Substance P and Neurokinin A Injections on Extracellular Dopamine Levels Measured with Microdialysis in the Striatum and Frontoparietal Cortex of Rats
Authors: Reid Malcolm S. , Herrera‐Marschitz Mario , Ungerstedt Urban
Published: October 2006

Extracellular levels of dopamine (DA) and its metabolite, 3,4‐dihydroxyphenylacetic acid (DOPAC), in the striatum and frontoparietal (sensorimotor) cortex in halo‐ thane‐anesthetized rats were analyzed simultaneously using in vivo microdialysis. Basal DA levels, measured from the microdialysis perfusate, were 6.4 ± 0.8 nM (n = 15) in the striatum and 0.9 ± 0.1 nM (n = 15) in the frontoparietal cortex. Subcutaneous injections of d‐amphetamine (2 mg/kg) increased DA levels 10‐fold in the striatum and fivefold in the cortex. Injections of substance P (0.07 nmol/0.2 μl) into the substantia nigra pars reticulata (SNR) increased DA and DOPAC levels ∼30% in the ipsilateral striatum and 50% in the ipsilateral frontoparietal cortex. Injections of neurokinin A (0.09 nmol/0.2 μl) into the SNR increased DA and DOPAC levels ∼30% in the ipsilateral striatum but did not significantly affect DA levels in the ipsilateral frontoparietal cortex, although DOPAC levels were increased by ∼50%. It is suggested that striatal and cortical DA release is regulated differently by nigral substance P and neurokinin A terminals.