bluuebunny/crossref_metadata_2025 huggingface dataset:
Ca2+ Regulates Hormone Secretion and Proopiomelanocortin Gene Expression in Melanotrope Cells via the Calmodulin and the Protein Kinase C Pathways
Authors: Loeffler J. Ph. , Kley N. , Louis J. C. , Demeneix B. A.
Published: October 2006

Abstract The mechanism by which Ca2+ regulates proopiomelanocortin (POMC)‐derived peptide secretion and POMC mRNA levels was investigated in primary cultures of porcine intermediate lobe (IL) cells maintained in serum‐free medium. POMC gene expression was evaluated by the dot blot hybridization assay with a 32P‐labeled DNA probe complementary to the full‐length sequence of porcine POMC mRNA. Treatment of IL cells for 24 h with the calmodulin (CAM) antagonists W7 and W13 reduced POMC mRNA levels by a maximum of 50% in a dose‐dependent manner (ED50± 10‐8M). Accumulation of α‐melanocyte‐stimulating hormone (α‐MSH) in the medium was also depressed by 50% after 8 h of treatment. The role of protein kinase C (PKC) was investigated by depleting the IL cell PKC content with phorbol ester treatment. Phorbol 12‐myristate 13‐acetate (PMA) at 5 X 10‐8M induced a rapid translocation of cytoplasmic PKC activity toward the membrane. After 12 h of PMA treatment, PKC activity was undetectable in either the cytoplasmic or the particulate fractions. The same dose of PMA induced a time‐dependent decrease in POMC mRNA levels (50% inhibition after 24 h). The same effect was seen with the phorbol ester phorbol 12,13‐dibutyrate at 5 X 10‐8M, whereas the inactive phorbol ester 4α‐phorbol at 5 X 10‐8M was without effect after 24 h of treatment. PMA treatment had a biphasic effect on α‐MSH secretion. After 8 h of PMA treatment, peptide accumulation in the medium was significantly increased, whereas a 24‐h treatment with PMA inhibited α‐MSH secretion as compared with untreated controls. Treatments with neither CAM antagonists nor phorbol esters decreased [3H]uridine incorporation into trichloroacetic acid‐precipitable material, results suggesting that the observed effects on gene expression are limited to POMC or to a restricted number of genes. Taken together, these results suggest that in the IL of the pituitary, Ca2+ modifies hormone secretion and POMC gene expression via the CAM and PKC pathways.
Inositol Phospholipid Metabolism During and Following Synaptic Activation: Role of Adenosine
Authors: Rubio R. , Bencherif M. , Berne R. M.
Published: October 2006

Abstract: The metabolic pathway of inositol phospholipids represents a series of synthetic and hydrolytic reactions with inositol as a by‐product. Hence, the rate of [3H]inositol release from prelabeled phospholipids can be used as a reflection of activity of this pathway. In the frog sympathetic ganglion prelabeled with [3H]inositol, we studied the effect of synaptic activity (orthodromic stimulation) on release of 3H‐label into the medium. This release was interpreted as [3H]inositol release. The value was low at rest and increased significantly by 32% during orthodromic stimulation (20 Hz for 5 min). However, on cessation of the stimulation, [3H]inositol release increased rapidly by 148% and remained elevated for at least 45 min. This increase in [3H]inositol release during and after the stimulation period was reduced by suffusion of the ganglia with adenosine. We hypothesized that synaptic activation releases a long‐lasting stimulatory agonist and a short‐lasting inhibitory (adenosine) agonist or agonists affecting [3H]inositol release. To demonstrate the presence of a stimulatory agonist, two sympathetic ganglia were used. One was prelabeled with [3H]inositol, and the other was not. The two ganglia were placed together in a 5‐μl droplet of Ringer's solution containing atropine. Orthodromic stimuli applied to the nonlabeled ganglion elicited release of [3H]inositol from the nonstimulated ganglion. To test whether the adenosine formed during orthodromic stimulation inhibits [3H]inositol release, we destroyed endogenous adenosine by suffusion of the ganglia with adenosine deaminase during the stimulation period. We found that adenosine deaminase induced large increases in [3H]inositol release during the stimulation period, in contrast to an increase seen only during the poststimulation period when adenosine deaminase was omitted. Because [3H]inositol release is assumed to parallel changes in content of inositol phosphates, we anticipated no changes of the levels of these compounds during orthodromic stimulation. However, measurements showed that levels of inositol phosphates and inositol phospholipids were all elevated except for phosphatidylinositol 4‐phosphate. On termination of the stimulus, they remained elevated, with a further increase in levels of inositol trisphosphate and phosphatidylinositol 4‐phosphate. We conclude that endogenous adenosine inhibits [3H]inositol release, possibly by modulating several of the steps of the inositol phospholipid pathway. The various alternatives are discussed.
AH5183 and Cetiedil: Two Potent Inhibitors of Acetylcholine Uptake into Isolated Synaptic Vesicles from Torpedo marmorata
Authors: Diebler Marie‐Françoise , Gaudry‐Talarmain Yvette Morot
Published: October 2006

Abstract: Synaptic vesicles purified on a sucrose‐KCl sedimentation gradient were tested for their ability to accumulate [l‐14C]acetylcholine ([l‐14C]ACh) in the absence and in the presence of AH5183 and cetiedil. Kinetic studies of ACh transport showed that it was time dependent and saturable as a function of ACh concentration, with a KT of 1.2 mM. The protein‐modifying agents N‐ethylmaleimide and 4‐chloro‐7‐nitrobenzo‐2‐oxa‐1,3‐diazole were powerful inhibitors of ACh uptake. In agreement with other studies, AH5183 was found to be a potent inhibitor of ACh uptake by synaptic vesicles. Inhibition was of the mixed noncompetitive type, and the inhibition constant was 45.2 ± 3.4 nM. Cetiedil, a drug that resembles ACh, was previously shown on intact nerve endings to inhibit the translocation of newly synthesized ACh into the synaptic vesicle compartment, and we demonstrate here that cetiedil is indeed an efficient blocker of ACh uptake by isolated synaptic vesicles. It acted as a competitive inhibitor, with a Ki of 118.5 ± 9.5 nM. Neither ATP‐dependent calcium uptake nor Mg2+‐ATPase activity was affected by the drugs, a finding showing their specificity toward the ACh uptake process. The binding of L‐[3H]AH5183 to intact vesicles was characterized in the absence or the presence of ACh or cetiedil. Saturation experiments showed a total binding capacity of ∼ 126 pmol/mg of vesicular protein and a dissociation constant of 19.9 ± 4.1 nM under control conditions. Whereas the specific binding of l‐[3H]AH5183 remained unaffected by ACh, even at high concentrations, cetiedil was shown to act as a competitive inhibitor with a Ki of 4.6 μM. We conclude that both AH5183 and cetiedil are efficient inhibitors of ACh transport by isolated synaptic vesicles but that they are likely to act at different sites.
Regulation of Thyrotropin Releasing Hormone Degrading Enzymes in Rat Brain and Pituitary by L‐ 3,5,3′‐Triiodothyronine
Authors: Suen Chen‐Shian , Wilk Sherwin
Published: October 2006

Abstract: The effect of treatment with L‐3,5,3′‐triiodothyro‐nine (T3) on the levels of pyroglutamyl peptidase I and pyroglutamyl peptidase II in rat brain regions, pituitary, and serum was studied. Pyroglutamyl peptidase I cleaves pyroglutamyl peptides such as thyrotropin releasing hormone (TRH), luteinizing hormone releasing hormone, neurotensin, and bombesin, whereas pyroglutamyl peptidase II appears to be specific for TRH. Acute administration of T3 did not affect pyroglutamyl peptidase I in any of the regions studied, whereas pyroglutamyl peptidase II was significantly elevated in frontal cortex and pituitary. Treatment with T3 for 10 or 14 days significantly elevated pyroglutamyl peptidase I in pituitary, hypothalamus, olfactory bulb, hippocampus, and thalamus. Chronic T3 treatment elevated pyroglutamyl peptidase II in frontal cortex and in serum. These studies demonstrate regulation of neuropeptide degrading enzymes by thyroid hormones in vivo. This regulation may play a role in the negative feedback control of thyroid status by T3.
Regulation of the Vesamicol Receptor in Cholinergic Synaptic Vesicles by Acetylcholine and an Endogenous Factor
Authors: Noremberg Krystyna , Parsons Stanley M.
Published: October 2006

Abstract: Cholinergic synaptic vesicles obtained from Torpedo electric organ have an active transport system for acetylcholine (ACh). Linked to ACh transport is a cytoplasmically oriented receptor for the inhibitory drug (–)‐trans ‐ 2‐(4‐phenylpiperidino)cyclohexanol (vesamicol, formerly AH5183). Storage of freshly isolated vesicles for several days leads to more vesamicol binding. This can be induced immediately by hyposmotic lysis of the vesicles, which reseal to form right‐side‐out ghosts. The increased drug binding was due to a twofold increase in the affinity and a 20% increase in the amount of the receptor expressed, probably as a result of the release of an endogenous factor. Binding of vesamicol to ghosts was specifically inhibited by exogenous ACh acting with a dissociation constant of 18 mM. This suggests that the vesamicol binding site probably is linked to a low‐affinity ACh binding site that is different from the higher affinity transport binding site. Equilibrium and kinetic attempts to determine whether exogenous ACh acts on the outside or the inside of the ghost membrane to inhibit vesamicol binding failed because of rapid equilibration of exogenous ACh across the ghost membrane. It is argued that the endogenous factor released by hyposmotic lysis might be ACh. Potential roles for such a transmembrane signal regulating the vesamicol receptor are discussed.
Structural Specificity of Sugar Transport at the Blood‐Nerve Barrier
Authors: Rechthand Emanuel , Smith Quentin R. , Rapoport Stanley I.
Published: October 2006

Abstract: The major component of D‐glucose transfer across the membranous sites of the blood‐nerve barrier (BNB) occurs via a facilitative mechanism at a rate greater than twice the rate of D‐glucose metabolism by nerve. To characterize further properties of monosaccharide transport at the BNB, unidirectional transfer constant (K) values were determined in vivo in tibial nerve of anesthetized rats for radiolabeled mannitol, L‐glucose, and a series of D‐glucose analogs. K values (× 10−4 ml s−1 g−l) equaled 4.8 for 2‐deoxy‐D‐glucose, 3.7 for D‐glucose, 2.3 for 3‐O‐methyl‐D‐glucose, 1.4 for D‐man‐nose, 0.6 for D‐galactose, 0.2 for mannitol, and 0.19 for L‐glucose. The rank order of ratios between K values of a D‐hexose and D‐glucose, which reflects the rank order of affinity of the system for individual sugars, was 2‐deoxy‐D‐glucose > D‐glucose > 3‐O‐methyl‐D‐glucose > D‐mannose > D‐galactose. The results demonstrate that the order of substrate affinity of the monosaccharide carrier at the BNB is similar to that at cerebral capillaries and at erythrocytes. At normal concentrations of plasma D‐glucose, the contribution of simple passive diffusion to unidirectional D‐glucose influx across the BNB equals 5%, which is greater than that at cerebral capillaries and reflects the greater permeability to hydrophilic nonelectrolytes of the endoneurial vasculature.
Cyclic AMP‐Dependent Melatonin Production in Y79 Human Retinoblastoma Cells
Authors: Pierce Mary E. , Barker Diane , Harrington Julie , Takahashi Joseph S.
Published: October 2006

Abstract: Melatonin is rhythmically synthesized in some vertebrate retinas and has been implicated in the regulation of key rhythmic events in the photoreceptor‐pigment epithelial complex. In human retina, melatonin is present; however, no information exists on the cellular regulation of this hormone. We report here that the established human retinoblastoma cell line Y79 synthesizes and releases melatonin. Treatments that elevate cyclic AMP (cAMP) levels (forskolin, 8‐Br‐cAMP, and the phosphodiesterase inhibitor 3‐isobutyl‐1 ‐meth‐ylxanthine) all stimulate melatonin release from static cultures of Y79 cells. Other 8‐bromo nucleotide analogues (cyclic GMP, ATP, and AMP) are not effective. These results suggest that Y79 human retinoblastoma cells require a cAMP‐dependent mechanism for melatonin biosynthesis similar to that described previously in other vertebrates. This is the first demonstration of melatonin release from a cultured human cell line. These results support the idea that human retinal cells share homologies with pineal cells, as suggested by the condition trilateral retinoblastoma.
Subtype‐Selective Immunoprecipitation of the β2‐Adrenergic Receptor
Authors: Romano Carmelo , Williams William V. , Fischberg Daniel J. , Cocero Nanette , Weiner David B. , Greene Mark I. , Molinoff Perry B.
Published: October 2006

Abstract: Most antibodies known to interact with β‐adren‐ergic receptors do not exhibit subtype selectivity, nor do they provide quantitative immunoprecipitation. A monoclonal antibody, G27.1, raised against a synthetic peptide corresponding to the C‐terminus of the β2‐adrenergic receptor of hamster, is selective for the β2 subtype. G27.1 provides nearly quantitative immunoprecipitation of the β2‐acjrenergic receptor from hamster lung that has been photoaffinity‐labeled and solubilized with sodium dodecyl sulfate. Immunoprecipitation is completely blocked by nanomolar concentrations of the immunizing peptide. This antibody interacts with β2‐adrenergic receptors from three rodent species, but not with those from humans. When C6 glioma cells, which contain both β1 ‐ and β2‐adrenergic receptors, are photoaffinity‐labeled in the absence or presence of subtype‐selective antagonists, subtype‐selective photoaffinity‐labeling results. G27.1 can immunoprecipitate β‐, but not β1‐, adrenergic receptors from these cells. Similar results were obtained following subtype‐selective photoaffinity‐labeling of membranes from rat cerebellum and cerebral cortex. The β‐adrenergic receptors from C6 glioma cells and rat cerebral cortex exist as a mixture of two molecular weight species. These species differ in glycos‐ylation, as shown by endoglycosidase F digestion of crude and immunoprecipitated receptors.
Multiple Sclerosis Brain Immunoglobulins Stimulate Myelin Basic Protein Degradation in Human Myelin: A New Cause of Demyelination
Authors: de Rosbo Nicole Kerlero , Bernard Claude C. A.
Published: October 2006

Abstract: Membrane‐bound proteolysis may be implicated in the pathogenesis of demyelinating disorders including multiple sclerosis (MS). We previously found that the extent of myelin basic protein (MBP) degradation by the calcium‐activated neutral protease did not differ for isolated human control myelin or MS myelin. Hence we suggested that, if involved in demyelination, the myelin neutral protease must be activated in vivo by an increased availability of free calcium. The postulate was therefore tested that immunoglobulin (Ig) binding to myelin results in activation of the myelin neutral protease, possibly through release of free calcium from calcium‐binding sites of myelin. Isolated myelin from the brains of controls and patients with MS were incubated with purified Igs eluted from the brains of patients with MS or controls and degradation of MBP was assessed by quantitative electroimmunoblotting. Such degradation was significantly greater in myelin incubated in the presence of MS Igs than in myelin incubated without added Igs or in the presence of control Igs. Furthermore, the degree of MBP degradation in myelin incubated with control Igs was similar to that observed in myelin incubated without added Igs. Accordingly, it is suggested that Ig in MS brain potentiates myelin breakdown. Moreover activation of membrane‐bound proteolysis by Ig binding to myelin appears to represent a hitherto undescribed pathway for demyelination in MS.
Inactivation of G1ucose‐6‐Phosphate Dehydrogenase Isozymes from Human and Pig Brain by Aluminum
Authors: Cho Sung‐Woo , Joshi J. G.
Published: October 2006

Abstract: Prolonged intake of low levels of aluminum from the drinking water has been found to increase the aluminum content in rat brain homogenates and to reduce the activity of hexokinase and glucose‐6‐phosphate dehydrogenase (G6PD). To determine the interaction of G6PD with aluminum in the brain, we have recently purified two isozymes of G6PD (isozymes I and II) from human and pig brain. Unlike isozyme I, isozyme II also had 6‐phosphogluconate dehydrogenase (6‐PGD) activity. We report here that G6PD isozymes I and II from human and pig brain purified to apparent homogeneity are inactivated by aluminum. Aluminum did not affect the 6‐PGD activity of isozyme II. The aluminum‐inactivated enzyme contained 1 mol of aluminum/mol of enzyme subunit. The protein‐bound metal ion was not dissociated by exhaustive dialysis at 4°C against 10 mM Tris‐HCI (pH 7.0) containing 0.2 mM EDTA. Preincubation of aluminum with citrate, NADP+, EDTA, NaF, ATP. and apotransferrin protected the G6PD isozymes against aluminum inactivation. However, when the G6PD isozymes were completely inactivated by aluminum, only citrate, NaF, and apotransferrin restored the enzyme activity. The dissociation constants for the enzyme‐aluminum complex of the isozymes varied from 2 to 4 μM, as measured by using NaF, a known chelator for aluminum. Inhibition of G6PD by low levels of aluminum further strengthens the suggested role of aluminum toxicity in the energy metabolism of the brain.
Inositol Hexakisphosphate (Phytic Acid) Enhances Ca2+Influx and D‐[3H]Aspartate Release in Cultured Cerebellar Neurons
Authors: Nicoletti Ferdinando , Bruno Valeria , Fiore Lucia , Cavallaro Sebastiano , Canonico Pier Luigi
Published: October 2006

Abstract: Inositol hexakisphosphate (InsP6) increased 45Ca2+uptake in cultured cerebellar granule cells. This increase was concentration dependent (EC50= 20 μM), exhibited slow kinetics, and was present after 5 days of cell maturation in culture. InsP6 also enhanced D‐[3H]aspartate release in cerebellar granule cells at 11‐12 days in vitro. Stimulation of 45Ca2+ uptake was also produced by inositol pentakisphos‐phate but not by inositol 1,3,4,5‐tetrakisphosphate. The increase in 45Ca2+ influx induced by InsP6 was independent of extracellular Na+ and was only partially reduced by the organic calcium channel blocker nifedipine. The intrinsic action of InsP6 was not affected by competitive or noncompetitive glutamate receptor antagonists. In addition, stimulations of 45Ca2+ uptake by InsP6 and glutamate were additive. These data provide evidence that InsP6 directly activates a specific population of neurons in the CNS.
GABAA Receptor Complex in an Experimental Model of Hepatic Encephalopathy: Evidence for Elevated Levels of an Endogenous Benzodiazepine Receptor Ligand
Authors: Basile Anthony S. , Gammal Sergio H. , Jones E. Anthony , Skolnick Phil
Published: October 2006

Abstract: The involvement of the γ‐aminobutyric acidA(GABAA) receptor complex in the pathogenesis of hepatic encephalopathy was examined in thioacetamide‐treated rats with fulminant hepatic failure. Partially purified extracts from encephalopathic rat brain were approximately three times more potent in inhibiting [3H]Ro 15‐1788 binding to benzodiazepine receptors than identically prepared extracts from control rats. High levels of inhibitory activity were also found in extracts of plasma, heart, and liver from thioacetamide‐treated rats. The inhibition of [3H]Ro 15‐1788 binding by brain extracts appeared to be competitive and reversible and was unaffected by treatment with either proteolytic enzymes or boiling. Further, GABA significantly enhanced the potency of these extracts in inhibiting [3H]flunitrazepam binding. In contrast, no differences were found in radioligand binding to the constituent recognition sites of the GABAA receptor complex in well‐washed brain membranes prepared from control and encephalopathic animals. These findings suggest that the recognition‐site qualities of the constituent proteins of the GABAA receptor complex are unchanged in an experimental model of hepatic encephalopathy. However, significant elevations in the level of a substance or substances with neurochemical properties characteristic of a benzodiazepine receptor agonist may contribute to the electrophysiological and behavioral manifestations of hepatic encephalopathy.
A Rapid Purification of L‐Glutamic Acid Decarboxylase from the Brain of the Locust Schistocerca gregaria
Authors: Stapleton Andrew , Tyrer N. Mark , Goosey Michael W. , Cooper Michael E.
Published: October 2006

Abstract: L‐Glutamic acid decarboxylase (GAD; EC 4.1.1.15) was purified to apparent homogeneity from the brain of the locust Schistocerca gregaria using a combination of chro‐matofocusing (Mono P) and gel filtration (Superose 12) media. The homogeneity of the enzyme preparation was established by native polyacrylamide gel electrophoresis (PAGE) with silver staining. The molecular weight of the purified enzyme was estimated from native gradient gel electrophoresis and gel filtration chromatography to be 97,000 ± 4,000 and 93,000 ± 5,000, respectively. When analysed by sodium do‐decyl sulphate‐PAGE, the enzyme was found to be composed of two distinct subunits of Mr 51,000 ± 1,000 and 44,000 ± 1,500. Tryptic peptide maps of iodinated preparations of these two subunits showed considerable homology, suggesting that the native enzyme is a dimer of closely related subunits. The purified enzyme had a pH optimum of 7.0‐7.4 in 100 mM potassium phosphate buffer and an apparent Km for glutamate of 5.0 mM. The enzyme was strongly inhibited by the carbonyl‐trapping reagent aminooxyacetic acid with an I50 value of 0.2 μM.
Role of Ribonuclease and Ribonuclease Inhibitor Activities in Alzheimer's Disease
Authors: Jones Linda M. , Knowler John T.
Published: October 2006

Abstract: It has been suggested that defects in the relationship between ribonuclease and its proteinaceous inhibitor could be a contributory factor in Alzheimer's disease. We have investigated this possibility further by analysing free and bound enzyme activities and the activity of the inhibitor in nine regions of diseased and normal brain. These were chosen to include areas known to be affected by the disease, regions not histologically affected but thought to be involved in the disease process, and areas not thought to be involved in the disease. Neither the enzyme nor its inhibitor is defective in its activities in the chosen areas of Alzheimer's disease brain when compared with those of carefully age‐matched controls
Lithium Reduqes the Accumulation or Inositol Polyphosphate Second Messengers Following Cholinergic Stimulation of Cerebral Cortex Slices
Authors: Kennedy Eleanor D. , John Challiss R. A. , Nahorski Stefan R.
Published: October 2006

Abstract: The ability of lithium to interfere with the metabolism of inositol phosphates in brain may underlie its therapeutic action in manic‐depressive illness. In these experiments, lithium, at therapeutic concentrations, enhanced the accumulation of [3H]inpsitol monophosphate but suppressed the accumulation of the putative second messengers [3H]inositol 1,4,5‐trisphosphate ([3H]Ins(1,4,5)P3) and f3H]inositol 1,3,4,5‐tetrakisphosphate following stimulation of cerebral cortex slices with carbachol. Mass measurements of Ins(1,4,5)P3showed similar inhibitory effects, which could be prevented by preincubation with myo‐inositol. These data may reveal the mechanism by which lithium can reduce polyphosphoinositide‐midiated neurotransmission in brain.
Effects of Lead Salts on the Uptake, Release, and Binding of γ‐Aminobutyric Acid: The Importance of Buffer Composition
Authors: Drew Colleen A. , Spence Ian , Johnston Graham A. R.
Published: October 2006

Abstract: The effects of lead on the uptake and release of γ‐[3H]aminobutyric acid ([3H]GABA) from rat brain slices were examined in solutions buffered with Tris‐HCl, sodium phosphate, and sodium bicarbonate. Lead acetate (10–250 μM) inhibited uptake and potassium‐stimulated release and facilitated spontaneous efflux only in solutions buffered with Tris‐HCl. Calcium‐independent binding of [3H]GABA was unaffected by lead acetate (1–100 μM) in Tris‐citrate buffer but was significantly inhibited by 3 μM lead acetate in Tris‐HCl solution. At the rat soleus neuromuscular junction, lead caused a dose‐dependent reduction of end‐plate potential amplitude at concentrations of 10–100 μM lead acetate in HEPES‐buffered solution but had no effect at these concentrations in phosphate‐buffered solution. Stability constants of lead complexes indicate that buffers containing carbonate and phosphate are unlikely to contain a significant concentration of Pb2+, as complexing by these anions would reduce the availability of free Pb2+. This study indicates that the choice of buffer is important when investigating the effects of lead on biological systems and that negative findings may result from the use of inappropriate buffers. It also has important clinical implications suggesting that some effects of lead poisoning may result from its ability to affect neurotransmitter systems directly and that local changes in pH and complexing anion concentrations in the CNS may influence its biological availability and, hence, variable biological responses.
Specific [3H]SCH23390 Binding to Dopamine D1 Receptors in Cerebral Cortex and Neostriatum: Role of Disulfide and Sulfhydryl Groups
Authors: Dewar Karen M. , Reader Tomás A.
Published: October 2006

Abstract: Receptor binding studies were performed in cerebral cortex (CTX) and neostriatum (CPU; caudate–putamen) using the dopamine D1 antagonist [3H]SCH23390. Because receptors are of protein nature, we examined the role of disulfide bonds (–SS–) and sulfhydryl groups (–SH) in the specific binding of [3H]SCH23390. Furthermore, membrane preparations contain a certain amount of lipid, so that treatments with –SH and –SS– reagents could determine whether the fixation of the radioligand was to protein or to the lipid moiety. Pretreatment of CTX and CPU membranes with dithioerythritol, l‐dithiothreitol, or 5,5′‐dithiobis(2‐nitro‐benzoic acid), as well as with the alkylating agent N‐ethyl‐maleimide, produced dose‐dependent decreases of specific [3H]SCH23390 binding in membrane preparations from both tissues. These changes were not reversible after up to two washes, but could be prevented in part if the treatments were performed in the presence of dopamine. Additional protection experiments were conducted with (+)‐ and (−)‐butaclamol, as well as with (+)‐ and (−)‐SKF38393. A series of saturation experiments (with pretreated membranes in the absence of reactives) demonstrated that the alkylation of –SS– groups reduced specific [3H]SCH23390 binding mainly through an affinity change, but l‐dithiothreitol and 5,5‐dithiobis(2‐nitrobenzoic acid) decreased the number of binding sites. The affinity of the receptor to agonists was examined with the two enantiomers of SKF38393; the inhibition curves showed that residual binding was not affected and stereospecificity was conserved. The present results provide evidence for the participation of both –SS– and –SH groups in the recognition site of the dopamine D1 receptor in both the CTX and the CPU.
Polychlorinated Convulsant Insecticides Potentiate the Protective Effect of NaCl Against Heat Inactivation of [3H]Flunitrazepam Binding Sites
Authors: Squires Richard F. , Saederup Else
Published: October 2006

Abstract: Six polychlorinated convulsant insecticides that potently inhibit t‐[35S]butylbicyclophosphorothionate ([35S]TBPS) binding to rat brain membranes also potentiate the protective effect of NaCl (200 mM) against heat inactivation of [3H]flunitrazepam binding sites on the same membranes. Similar effects were obtained with all “cage” convulsants tested. The rank order of potencies as heat protection potentiators was similar to the rank order of potencies as inhibitors of [35S]TBPS binding (α‐endosulfan > endrin > dieldrin > toxaphene > lindane). α‐Endosulfan and endrin are more potent in both respects than any previously reported picrotoxin‐like (cage) convulsant, but are much less toxic to mammals. The greatly reduced toxicities of α‐endosulfan and endrin in mammals may reflect partial γ‐aminobutyric acid (GABA)‐neutral properties of these compounds. Time courses of heat inactivation of [3H]flunitrazepam binding sites in the presence of 200 mM NaCl plus saturating concentrations of endrin or picrotoxin revealed monophasic components constituting about 88% of the binding sites, suggesting that virtually all [3H]flunitrazepam binding sites are coupled to picrotoxin binding sites in the GABA/benzodiazepine/picrotoxin receptor complex. Protection against heat inactivation constitutes a useful tool for characterizing the various allosterically linked binding sites in neurotransmitter receptor complexes.
In Vivo Labeling of Serotonin Uptake Sites with [3H]Paroxetine
Authors: Scheffel Ursula , Hartig Paul R.
Published: October 2006

Abstract: Previous work has shown that [3H]paroxetine is a potent and selective in vitro label for serotonin uptake sites in the mammalian brain. In the present study, [3H]paroxetine was tested in mice as an in vivo label for serotonin uptake sites. Maximum tritium concentration in the whole brain (1.4% of the intravenous dose) was reached 1 h after injection into a tail vein. Distribution of the tracer at 3 h after injection followed the distribution of serotonin uptake sites known from previous in vitro binding studies (r= 0.85). The areas of highest [3H]paroxetine concentration, in decreasing order, were: hypothalamus > frontal cortex > olfactory tubercles > thalamus > upper colliculi > brainstem > hippocampus > striatum > cerebellum. Preinjection of carrier paroxetine (1 mg/kg) significantly decreased [3H]paroxetine concentration in all areas except in the cerebellum, which is known to contain a relatively low number of specific binding sites. Kinetic studies showed highest specific [3H]paroxetine binding (tissue minus cerebellum) at 2 h after injection and slow clearance of activity thereafter (half‐time of dissociation from the hypothalamus, 215 min). The specificity of in vivo [3H]paroxetine binding was studied by preinjecting monoamine uptake blockers or receptor antagonists 5 min before administration of [3H]paroxetine. Serotonergic or muscarinic cholinergic receptor antagonists and dopamine or norepinephrine uptake blockers did not reduce the in vivo binding of [3H]paroxetine. In contrast, there was an excellent correlation (r= 0.99) between the in vivo inhibitory potencies of serotonin uptake blockers in this study and previously published in vitro data on inhibition of [3H]serotonin uptake in brain synaptosomes. In addition, [3H]paroxetine binding in the hypothalamus was found to be stereospecifically inhibited, (Z)‐norzimelidine displaying a greater than fourfold higher potency than the E isomer. These studies indicate that [3H]paroxetine is a potent and selective agent for the in vivo labeling of cerebral serotonin uptake sites.
Kainate Receptors in Xenopus Central Nervous System: Solubilisation with n‐Octyl‐β‐d‐Glucopyranoside
Authors: Henley Jeremy M. , Barnard Eric A.
Published: October 2006

Abstract: [3H]Kainate binding to membrane homogenates and detergent extracts prepared from Xenopus central nervous system was evaluated in 50 mM Tris‐citrate buffer, pH 7.0. In membrane fragment preparations, [3H]kainate bound with a KD of 54.4 nM to a large number of sites (Bmax= 27.8 pmol/mg of protein). Up to 80% of the total number of membrane‐bound binding sites were solubilised using the nonionic detergent n‐octyl‐β‐d‐glucopyranoside. Values for the KD of [3H]kainate for solubilised binding sites were 46.0 nM and 53.6 nM derived from equilibrium and kinetic binding experiments, respectively. Competitive binding studies revealed that a variety of ligands had similar Ki values in both membranes and solubilised extracts, with domoate and kainate being the most potent inhibitors of [3H]kainate binding. The dissociation rate of [3H]kainate from solubilised binding sites was 0.022 min–1. The binding component migrated in sucrose density gradients in a single 8.6S peak. These results demonstrate that the kainate receptor in Xenopus central nervous system, although similar to the [3H]kainate binding site from goldfish brain, differs in a number of important respects. In particular, the slower dissociation rate and higher affinity of [3H]kainate suggest that Xenopus provides the most convenient model system yet investigated for biochemical analysis of kainate receptors.
Nerve Growth Factor Potentiates the Hormone‐Stimulated Intracellular Accumulation of Inositol Phosphates and Ca2+ in Rat PC12 Pheochromocytoma Cells: Comparison with the Effect of Epidermal Growth Factor
Authors: van Calker Dietrich , Takahata Kyoya , Heumann Rolf
Published: October 2006

Abstract: The effects of nerve growth factor (NGF) and epidermal growth factor (EGF) on the intracellular accumulation of inositol phosphates and on cytosolic free Ca2+ concentrations were studied in rat PC12 pheochromocytoma cells. Both NGF and EGF potentiate in these cells the increase in the accumulation of inositol phosphates that is elicited by bradykinin and carbachol. A corresponding potentiation was also found for the agonist‐induced increase of cytosolic Ca2+ concentrations. The effect of NGF, but not that of EGF, is abolished when the cells are preincubated with 5′‐deoxy‐5′‐methylthioadenosine, an inhibitor of S‐adenosylhomocysteine hydrolase. These results suggest that an increased response to hormones, which act via phosphoinositide‐derived second messengers, may be important in the mechanism of action of NGF and EGF.
Characterization of Cysteine Proteases Functioning in Degradation of Dynorphin in Neuroblastoma Cells: Evidence for the Presence of a Novel Enzyme with Strict Specificity Toward Paired Basic Residues
Authors: Satoh Mitsuo , Yokosawa Hideyoshi , Ishii Shin‐ichi
Published: October 2006

Abstract: Two dynorphin‐degrading cysteine proteases, I and II, were extracted with Triton X‐100 from neuroblastoma cell membrane, isolated from accompanying dynorphin‐degrading trypsin‐like enzyme by affinity chromatography on columns of soybean trypsin inhibitor‐immobilized Sepharose and p‐mercuribenzoate–Sepharose, and separated by ion‐exchange chromatography on diethylaminoethyl (DEAE)‐cellulose and TSK gel DEAE‐5PW columns. Cysteine protease II was purified further by hydroxyapatite chromatography and gel filtration. The molecular weights of cysteine proteases I and II were estimated to be 100,000 and 70,000, respectively, by gel filtration. Both of the enzymes were inhibited by p‐chloromercuribenzoate, N‐ethylmaleimide, and high‐molecular‐weight kininogen, but not or only slightly inhibited by diisopropylphosphorofluoridate, antipain, leupeptin, E‐64, calpain inhibitor, and phosphoramidon. Cysteine protease I cleaved dynorphin(1–17) at the Arg6‐Arg7 bond with the optimum pH of 8.0, whereas II cleaved dynorphin(1–17) at the Lys11‐Leu12 bond and the Leu12‐Lys13 bond with the optimum pH values of 8.0 and 6.0, respectively. These bonds corresponded to those that had been proposed as the initial sites of degradation by neuroblastoma cell membrane. Cysteine protease I was further found to show strict specificity toward the Arg‐Arg doublet, when susceptibilities of various peptides containing paired basic residues were examined as substrates for the enzyme.
Developmental Expression of the Glial Fibrillary Acidic Protein mRNA in the Central Nervous System and in Cultured Astrocytes
Authors: Tardy M. , Fages C. , Riol H. , Le Prince G. , Rataboul P. , Charriere‐Bertrand C. , Nunez J.
Published: October 2006

Abstract: The expression of glial fibrillary acidic protein (GFAP)‐mRNA during mouse brain development and in astroglial primary cultures has been investigated by using two approaches: Northern‐blot evaluation using a specific cDNA probe, and cell‐free translation associated with immunoprecipitation. During brain maturation (4–56 days postnatal), the GFAP‐mRNA underwent a biphasic evolution. An increase was observed between birth and day 15 (i.e., during the period of astroglial proliferation), which was followed by a decrease until day 56 (i.e., during astroglial cell differentiation). At older stages (300 days), an increase was observed, which might reflect gliosis. During astroglial in vitro development (7–32 days in culture), the GFAP‐mRNA showed similar variations. An increase, observed during the period of astroglial proliferation (7–18 days), was followed by a decrease which occurred in parallel to marked changes in cell shape, cell process outgrowth, and the organization and accumulation of gliofilaments. During the same culture period (7–32 days), α‐tubulin mRNA, which was used as an internal standard, did not vary significantly. These results show that the increase of the GFAP protein and of gliofilaments observed both in vivo and in vitro during astroglial differentiation cannot be ascribed to an accumulation of the GFAP‐mRNA. It might be that more than one mechanism regulates the levels of free and polymerized GFAP and of its encoding mRNA.
Metalloendoprotease Inhibitors Block Fast Axonal Transport
Authors: Hammerschlag Richard , Bolen Franci A. , Stone George C.
Published: October 2006

Abstract: Metalloendoprotease activity that was sensitive to the metal chelator 1,10‐phenanthroline and to synthetic dipeptide substrates of the enzyme was detected in homogenates of dorsal root ganglia (DRG) and spinal nerve from the bullfrog. Exposure of an intact in vitro preparation of DRG and spinal nerves to 1,10‐phenanthroline led to a dose‐dependent depression in the accumulation of fast‐transported 3H‐labeled protein proximal to a nerve ligature. In nonligated preparations, the chelator treatment reduced the amount of transported protein entering the nerve; no marked effect on the transport rate was observed. Exposure of a desheathed region of spinal nerve to 1,10‐phenanthroline, while DRG were maintained in control medium, resulted in a slight depression of fast transport. This effect was not dose dependent over the range that produced a dose response when both DRG and spinal nerve were exposed to the drug. Treatment of DRG and spinal nerve with the metalloendoprotease substrate analogues carbobenzoxy (CBZ)‐Ser‐Leu‐amide or CBZ‐Gly‐Leu‐amide inhibited fast axonal transport, whereas treatment with CBZ‐Gly‐Gly‐amide, which is not a substrate, had no detectable effect on transport. Selective exposure of desheathed nerve trunk to CBZ‐Ser‐Leu‐amide inhibited fast transport, but the effect was less marked than when DRG and nerve trunk were treated. Although previous studies have focused on the role of metalloendoprotease activity in exocytosis, the present data suggest that the enzyme may also be involved in earlier stages of intracellular transport.
Regulatory Properties of Calcium/Calmodulin‐Dependent Protein Kinase II in Rat Brain Postsynaptic Densities
Authors: Rich Devra P. , Cdlbran Roger J. , Schworer Charles M. , Soderling Thomas R.
Published: October 2006

Calcium/calmodulin (CaM)‐dependent protein kinase II (CaM‐kinase II) contained within the postsynaptic density (PSD) was shown to become partially Ca2+‐indepen‐dent following initial activation by Ca2+/CaM. Generation of this Ca2+‐independent species was dependent upon auto‐phosphorylation of both subunits of the enzynme in the presence of Mg2+/ATP/Ca2+/CaM and attained a maximal value of 74 ± 5% of the total activity within 1–2 min. Subsequent to the generation of this partially Ca2+‐independent form of PSD CaM‐kinase II, addition of EGTA to the autophos‐phorylation reaction resulted in further stimulation of 32PO4 incorporation into both kinase subunits and a loss of stimulation of the kinase by Ca2+/CaM. Examination of the sites of Ca2+‐dependent autophosphorylation by phosphoamino acid analysis and peptide mapping of both kinase subunits suggested that phosphorylation of Thr286/287 of the α‐ and β‐subunits, respectively, may be responsible for the transition of PSD CaM‐kinase II to the Ca2+‐independent species. A synthetic peptide 281–309 corresponding to a portion of the regulatory domain (residues 281–314) of the soluble kinase inhibited syntide‐2 phosphorylation by the Ca2+‐independent form of PSD CaM‐kinase II (IC50= 3.6 ± 0.8 μM). Binding of Ca2+/CaM to peptide 281–309 abolished its inhibitory property. Phosphorylation of Thr286 in peptide 281–309 also decreased its inhibitory potency. These data suggest that CaM‐kinase II in the PSD possesses regulatory properties and mechanisms of activation similar to the cytosolic form of CaM‐kinase II.
Detection of Synenkephalin, the Amino‐Terminal Portion of Proenkephalin, by Antisera Directed Against Its Carboxyl Terminus
Authors: Stell William K. , Chaminade Michel , Metters Kathleen M. , Rougeot Catherine , Dray Fernand , Rossier Jean
Published: October 2006

Synenkephalin (SYN), the nonopioid amino‐terminal portion of proenkephalin (PRO), is stable and well conserved in mammals and therefore a promising marker for PRO systems. We immunized rabbits with synthetic [Tyr63]SYN(63–70)‐octapeptide, coupled by glutaraldehyde to bovine serum albumin. In radioimmunoassay (RIA) using antiserum no. 681, [Tyr63]SYN(63–70)‐octapeptide as standard, and 125I‐[Tyr63]SYN(63–70)‐octapeptide as tracer, the IC50 was ∼51 fmol/100‐μl sample at equilibrium or 12 fmol/100 μl in disequilibrium, and the sensitivity was ∼3 fmol/100 μl. Cross‐reactivity of the assay was 100% with [Cys63]SYN(63–70)‐octapeptide and with bovine adrenal 8.6‐kilodalton peptide digested with trypsin and carboxypeptidase B, but < 0.1% with transforming growth factor‐alpha, 2 × 10‐6 with Leu‐Leu‐Ala [SYN(68–70)‐tripeptide], and ≪ 10‐6 with all other peptides tested. Therefore in RIA this antiserum is specific for the free carboxyl terminus of SYN. Because the peptide detected after enzyme digestion is the complete SYN(63–70)‐octapeptide, we refer to the RIA as an assay for SYN(63–70). Tissue extracts were made in 1 M acetic acid, dried, reconstituted in Tris‐CaCl2, and digested sequentially with trypsin plus carboxypeptidase B. Extracts from bovine corpus striatum gave SYN(63–70) RIA dilution curves parallel to the standard curve both before and after digestion. Digestion increased the amount of immunoreactive SYN(63–70) in striatum by a factor of 1.5–2.0. The ratio of total immunoreactive [Met5]enkephalin to total immunoreactive SYN(63–70) (after sequential digestion) was ∼6:1. At least 90% of the immunoreactive SYN(63–70) in extracts of bovine caudate nucleus eluted from Sephadex G‐100 with an apparent molecular weight equal to that of bovine PRO(1–77). Using the new RIA we were able to detect and characterize SYN processing for the first time in extracts of whole rat brain, human globus pallidus, and human pheochromocytoma. Results in these tissues were similar to those in cattle, in that most stored SYN had been processed to a free carboxyl terminus. Since the C‐terminal octapeptide of SYN is practically identical in all known mammalian PRO, antiserum no. 681 should be useful for detecting, measuring, and purifying SYN from various mammals, including human beings.
Binding of Progesterone to Nerve Cell Membranes of Rat Brain Using Progesterone Conjugated to 125I‐Bovine Serum Albumin as a Ligand
Authors: Ke Ferng‐Chun , Ramirez Victor D.
Published: October 2006

Radioiodinated bovine serum albumin conjugated to progesterone was used as a probe to examine binding parameters of steroids to membrane preparations from rat brain tissue. The binding of 11α‐hydroxyprogesterone‐11‐hemisuccinate‐125I‐bovine serum albumin conjugate reached saturation after 30 min of incubation at 5°C. Several bovine serum albumin‐conjugated steroids were then tested for competition displacement studies. Among these steroid conjugates, the bovine serum albumin conjugate at position 3 of progesterone had the highest affinity, with an estimated inhibition constant of 28.5 ± 2.1 nM (n = 3), whereas bovine serum albumin itself and the 17β‐estradiol 6‐(O‐carboxy‐methyl)oxime‐bovine serum albumin conjugate showed no specific displacement. In addition, the binding sites were localized in an axolemma‐enriched fraction of rat brainstem. Specific binding was obtained in tissues from cerebral cortex, brainstem, cerebellum, corpus striatum, and hypothalamus, but little or no binding occurred in uterus, ovary, liver, and spleen. The present data indicate that progesterone‐125I‐bovine serum albumin conjugate can be used as a ligand to study progesterone‐membrane receptor interactions.
2′‐Deoxycoformycin Inhibition of Adenosine Deaminase in Rat Brain: In Vivo and In Vitro Analysis of Specificity, Potency, and Enzyme Recovery
Authors: Padua R. , Geiger J. D. , Dambock S. , Nagy J. I.
Published: October 2006

2′‐Deoxycoformycin (DCF), a potent inhibitor of adenosine deaminase (ADA), is increasingly used as a tool to investigate adenosine metabolism and neuromodulation. To advance further the usefulness of DCF for studies of purines in the CNS, we determined the inhibitory potency of this compound against ADA and adenylate deaminase (AMPDA) in brain, the rate of ADA recovery in various brain regions after single or repeated intraperitoneal DCF administrations, and the effect of DCF on several neurotransmitter synthetic enzymes. In vitro, the Ki values for inhibition of ADA and AMPDA were found to be 23 pM and 233 μM, respectively. In vivo, DCF inhibited ADA with ED50 values ranging from 155 to 280 μg/kg at 2 h posttreatment, and 98% inhibition was achieved with 1 mg/kg. AMPDA activity was not affected by doses up to 5.0 mg/kg. In contrast to the >95% inhibition of ADA seen 1 day after DCF at 5 mg/kg, the effectiveness of a second similar DCF treatment on the activity that had recovered by 14 days was dramatically reduced. Eight days after DCF treatment with doses of 5–50 mg/kg, the degree of ADA activity recovery in 10 brain regions examined was similar; it averaged 35% of control values at the low dose but showed some heterogeneity, ranging from 15 to 54% of control values, at the higher doses. Forty days after treatment with a single dose of 5 mg/kg, ADA activity recovered by 68–78% of control values in brain regions with normally high levels of activity and by 44–59% of control values in other regions. The activities of choline acetyltransferase, glutamic acid decarboxylase, and histidine decarboxylase (an enzyme colocalized with ADA in hypothalamic neurons) were unaffected by DCF treatment, a result suggesting the lack of a generalized neurotoxic effect. The very low doses of DCF required for ADA inhibition in vivo are consistent with the high potency of this drug against ADA in vitro, and any physiological effects observed at low doses might therefore be ascribed to inhibition of ADA.