Effect of aminooxyacetic acid on the release of preloaded [3H]GABA and radioactive metabolites from slices of developing mouse brain

The release of [³H]-aminobutyric acid (GABA) and its radioactive metabolites from slices of the cerebral cortex, cerebellum, striatum and brain stem of developing and adult mice was studied. The slices were incubated and superfused in the absence and presence of the GABA aminotransferase (GABA-T) inhibitor aminooxyacetic acid (AOAA). Exposure to 100 M AOAA totally inhibited GABA-T and all radioactivity released from slices was in authentic GABA. In studies on developing brain the 10-M concentration was also effective enough, except in cerebellar slices. In the absence of AOAA the major part of radioactivity spontaneously released from slices of adult cerebral cortex and cerebellum was tritiated water and still about one third part in the presence of 10 M AOAA. Potassium stimulation induced only the release of radioactive GABA but not labeled metabolites in both presence and absence of AOAA. AOAA reduced the stimulation-induced release of GABA. It is recommended that the use of GABA-T inhibitors should be discontinued in release experiments. Then labeled GABA must be separated in the effluents from its radioactive breakdown products.     

The Catalytic Transition State in ATP Synthase

The catalytic transition state of ATP synthase has been characterized and modeled by combined use of (1) Mg-ADP–fluoroaluminate, Mg-ADP–fluoroscandium, and corresponding Mg-IDP–fluorometals as transition-state analogs; (2) fluorescence signals of -Trp331 and -Trp148 as optical probes to assess formation of the transition state; (3) mutations of critical catalytic residues to determine side-chain ligands required to stabilize the transition state. Rate acceleration by positive catalytic site cooperativity is explained as due to mobility of -Arg376, acting as an arginine finger residue, which interacts with nucleotide specifically at the transition state step of catalysis, not with Mg-ATP- or Mg-ADP-bound ground states. We speculate that formation and collapse of the transition state may engender catalytic site / subunit-interface conformational movement, which is linked to -subunit rotation.     

GABA synthesis in brain slices is dependent on glutamine produced in astrocytes

The rate of -aminobutyric acid (GABA) synthesis in rat-brain slices was determined by inhibiting GABA transaminase with 20-M gabaculine and measuring the increase of GABA. Added 500-M glutamine increased the rate of GABA synthesis by 50%, indicating that glutamate decarboxylase is not saturated in brain slices. The stimulation of GABA synthesis with added glutamine in brain slices was much less than that reported for synaptosomes. The lower stimulation in slices was attributable to astrocytic glutamine production, as the rate of GABA synthesis decreased by 44% when glutamine production was inhibited with methionine sulfoximine. Added glutamine restored the rate to the maximal value observed in brain slices. The rate of GABA synthesis was decreased by 65% in slices pretreated with an inhibitor of glutaminase, and added glutamine did not reverse this effect. These results suggest that glutamine produced by astrocytes is a quantitatively important precursor of GABA synthesis in cortical slices.     

Structural and functional studies of the nicotinic acetylcholine receptor by solid-state NMR

Over the last seven years, solid-state NMR has been widely employed to study structural and functional aspects of the nicotinic acetylcholine receptor. These studies have provided detailed structural information relating to both the ligand binding site and the transmembrane domain of the receptor. Studies of the ligand binding domain have elucidated the nature and the orientation of the pharmacophores responsible for the binding of the agonist acetylcholine within the agonist binding site. Analyses of small transmembrane fragments derived from the nicotinic acetylcholine receptor have also revealed the secondary structure and the orientation of these transmembrane domains. These experiments have expanded our understanding of the channels structural properties and are providing an insight into how they might be modulated by the surrounding lipid environment. In this article we review the advances in solid-state NMR applied to the nicotinic acetylcholine receptor and compare the results with recent electron diffraction and X-ray crystallographic studies.Presented at the Biophysical Society Meeting on Ion channels – from structure to disease held in May 2003, Rennes, France     

Movement of thallous ion across the ascites cell membrane

Summary The movement of thallous ion (Tl⁺ across the ascites cell membrane has been characterized. Analogous to previous findings for⁸⁶Rb⁺ (used as a tracer for K⁺),²⁰⁴Tl⁺-influx could be resolved into three components: a ouabain-inhibitable pump flux, a passive flux, and a furosemide- or NO ₃ ^– -sensitive exchange flux. Although Tl⁺ moved approximately nine times faster across the membrane than K⁺, the pump/leak ratio was equal for the two ions. This suggests that the pump- and leak-pathways share a common rate-limiting step. The exchange mechanism was shown to provide close coupling between the Tl⁺- and K⁺-gradients.     

β-Dl-Methylene-aspartate,an inhibitor of aspartate aminotransferase,potently inhibitsl-glutamate uptake into astrocytes

[³H]Glutamate uptake into astrocytes in primary culture was potently inhibited by the aspartate analoguesl- andd-aspartic acid,Dl-threo--hydroxy-aspartic acid,l-aspartic acid--hydroxymate (IC50's: 136, 259, 168, and 560 M, respectively) and by -Dl-methylene-aspartate, a suicide inhibitor of asparate aminotransferase (IC50: 524 M), and by the endogenous sulphur-containing amino acidl-cysteinesulfinic acid (IC50: 114 M). [³H]Glutamate uptake was not significantly affected by either N-methyl-d-aspartate orDl-homocysteine thiolactone. These results demonstrate that other excitatory amino acids including aspartate andl-cysteinesulfinic acid (but excludingl-homocysteic acid) interact with the glutamate transport system of astrocytes. Inhibition of glutamate uptake may significantly increase the level of neuronal excitability.     

Pharmacology of the neuronal nicotinic acetylcholine receptor of cultured Kenyon cells of the honeybee, <Emphasis Type="Italic">Apis mellifera</Emphasis>

We investigated the pharmacology of the nicotinic acetylcholine receptor of honeybee Kenyon cells, a subset of olfactory interneurons, which are crucial for olfactory learning and memory. Whole-cell currents were recorded using patch-clamp techniques. Pressure application of agonists induced inward currents in cultured Kenyon cells at holding potentials of –110 mV. Acetylcholine or carbamylcholine were full agonists, nicotine, epibatidine and cytisine were only partial agonists. Coapplications of these partial agonists with acetylcholine reduced the current amplitude. The most efficient antagonists were dihydroxy--erythroidine (EC₅₀=0.5 pmol·l^–1) and methyllycaconitine (EC₅₀=24 pmol·l^–1). The open channel blocker mecamylamine, d-tubocurarine and hexamethonium were rather weak blockers of the honeybee nicotinic response. Bath applications of the muscarinic antagonist atropine inhibited nicotinic currents dependent on concentration (EC₅₀=24.3 mol·l^–1). Muscarine, pilocarpine or oxotremorine (1 mmol·l^–1) did not induce any measurable currents. The non-cholinergic drugs strychnine, bicuculline and picrotoxin partially and reversibly blocked the acetylcholine-induced currents. Our results indicate the expression of only one nicotinic acetylcholine receptor subtype in cultured Kenyon cells. Muscarinic as well as non-cholinergic antagonists also inhibit the receptor function, distinguishing the honeybee nicotinic receptor from the typical nicotinic receptor of vertebrates and from many described insects receptors.     

Toxicity and neuronal transport of stable liposomes and phospholipid in the nervous system

When unilamellar stable liposomes composed of a dialkyl analog of phosphatidylcholine, tetradecyloctadec-11-eno(1)phosphocholine (dialkyl-PC), plus cholesterol at 11 molar ratio, and a trace of [³H]dialkyl-PC were injected into the vitreous of the rabbit eye, macrophage infiltration and phagocytosis of lipid were observed in retina including the epiretinal myelinated nerve fiber bundles, with no other neurotoxic effects. Little or no incorporation of [³H]dialkyl-PC was observed in the distal tissues of the optic system. With labile vesicles composed of egg lecithin, trace amounts of [³H]dialkyl-PC, and phosphatidic acid, no morphological changes occurred. After a lag of more than 7 days [³H]dialkyl-PC appeared in superior colliculus, indicating axonal transport of the lipid in an anterograde direction. Experiments with submandibular and parotid gland indicated retrograde transport of the lipid. The data do not suggest axonal transport of intact (stable) liposomes, but suggest that intact phospholipid molecules can be axonally transported.     

Action of nereistoxin on recombinant neuronal nicotinic acetylcholine receptors expressed in<Emphasis Type="Italic"> Xenopus laevis</Emphasis> oocytes

Nereistoxin (NTX), a natural neurotoxin from the salivary glands of the marine annelid worm Lumbriconereis heteropoda, is highly toxic to insects. Its synthetic analogue, Cartap, was the first commercial insecticide based on a natural product. We have used voltage-clamp electrophysiology to compare the actions of NTX on recombinant nicotinic acetylcholine receptors (nicotinic AChRs) expressed in Xenopus laevis oocytes following nuclear injection of cDNAs. The recombinant nicotinic AChRs investigated were chicken 7, chicken 42 and the Drosophila melanogaster/chicken hybrid receptors SAD/2 and ALS/2. No agonist action of NTX (0.1–100 M) was observed on chicken 7, chicken 42 and the Drosophila/chicken hybrid nicotinic AChRs. Currents elicited by ACh were reduced in amplitude by NTX in a dose-dependent manner. The toxin was slightly more potent on recombinant Drosophila/vertebrate hybrid receptors than on vertebrate homomeric (7) or heteromeric (42) nicotinic AChRs. Block by NTX of the chicken 7, chicken 42 and the SAD/2 and ALS/2 Drosophila/chicken hybrid receptors is in all cases non-competitive. Thus, the site of action on nicotinic AChRs of NTX, to which the insecticide Cartap is metabolised in insects, differs from that of the major nicotinic AChR-active insecticide, imidacloprid.     

Density functional and docking studies of retinoids for cancer treatment

The retinoic acid receptor (RAR) and retinoid X receptor (RXR) are members of the nuclear receptor superfamily. The ligand-binding domain contains the ligand-dependent activation function. The isotypes RAR, and are distinct pharmacological targets for retinoids involved in the treatment of various cancers and skin diseases. There is thus considerable interest in synthetic retinoids with isotype selectivity and reduced side effects. In this work we have focused on the retinoid acid receptor and three of its panagonists. We have carried out density functional geometry optimizations at the B3LYP/6-31G* level, computed two types of atomic charges and also electrostatic potentials. A docking program was used to investigate the interactions between the receptor and the three ligands. A theoretically more potent inhibitor, which was obtained by modifying one of the retinoic acids investigated, is proposed. Figure Superposition of the crystal structure (Å) of the 1FCX ligand with the proposed new inhibitor     

AKIN<Emphasis Type="Italic">β</Emphasis>3, a plant specific SnRK1 protein,is lacking domains present in yeast and mammals non-catalytic <Emphasis Type="Italic">β</Emphasis>-subunits

The SNF1/AMPK/SnRK1 heterotrimeric kinase complex is involved in the adaptation of cellular metabolism in response to diverse stresses in yeast, mammals and plants. Following a model proposed in yeast, the kinase targets are likely to bind the complex via the non-catalytic -subunits. These proteins currently identified in yeast, mammals and plants present a common structure with two conserved interacting domains named Kinase Interacting Sequence (KIS) and Association with SNF1 Complex (ASC), and a highly variable N-terminal domain. In this paper we describe the characterisation of AKIN3, a novel protein related to AKIN subunits of Arabidopsis thaliana, containing a truncated KIS domain and no N-terminal extension. Interestingly the missing region of the KIS domain corresponds to the glycogen-binding domain (-GBD) identified in the mammalian AMPK1. In spite of its unusual features, AKIN3 complements the yeast sip1sip2gal83 mutant. Moreover, interactions between AKIN3 and other AKIN complex subunits from A. thaliana were detected by two-hybrid experiments and in vitro binding assays. Taken together these data demonstrate that AKIN3 is a -type subunit. A search for -type subunits revealed the existence of 3-type proteins in other plant species. Furthermore, we suggest that the AKIN3-type subunits could be plant specific since no related sequences have been found in any of the other completely sequenced genomes. These data suggest the existence of novel SnRK1 complexes including AKIN3-type subunits, involved in several functions among which some could be plant specific.     

A propos des ≪chloride cells≫ dans l'épithélium des lamelles branchiales du poisson rouge

Résumé L'ultrastructure des lamelles branchiales et spécialement celle des chloride cells du poisson rouge (Carassius aureus) a été étudiée. Nous avons constaté que du matériel amorphe floconneux, faiblement adiélectronique était attaché aux endroits des creux apicaux. Afin de préciser la nature de ce matériel, nous avons étudié ces structures au microscope électronique avec les techniques suivantes: acide periodique méthènamine d'argent, colorations au fer colloïdal et au bleu d'alcian. Après la réaction à l'acide periodique méthènamine d'argent, de fines précipitations aux endroits des creux apicaux, correspondant au matériel floconneux visible après la fixation au glutaraldéhyde tétroxyde d'osmium, étaient visibles. La coloration au bleu d'alcian révélait des particules fortement colorées formant un film plus ou moins continu à la surface libre des lamelles, sauf aux endroits oò les chloride cells sont en contact avec la surface. Là et notamment dans les 2reux apicaux, du matériel légèrement granuleux, de faible densité, faisait une couche assez épaisse attachée à la membrane cellulaire. Tenant compte des résultats d'autres auteurs et de nos propres observations, nous considérons que la plus grande partie du matériel se trouvant à la surface des chloride cells, et particulièrement dans les creux apicaux, est de type glycoprotéique.

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The ultrastructure of the chloride cells in the gill epithelium of the goldfish

Summary The ultrastructure of the secondary lamellae of the gills and especially that of the chloride cells of Carassius aureus was studied. We found an amorphous, flakey, slightly adielectronic material in the areas of the apical pits. In order to determine the nature of this material, we studied these structures electronmicroscopically applying the periodic acid silver methenamine, colloidal iron and alcian blue methods. The periodic acid silver methenamine reaction, resulted in finely dispersed precipitations which were deposited in the areas of the apical pits and which correspond to the flakey material seen in the ordinary electron micrographs. The alcian blue method reveales strongly stained particles which form a more or less continuous film on the free surface of the lamellae, interrupted only at the level of the chloride cells. In these areas, notably within the apical pits, a rather thick layer of finely granular low-density material is attached to the plasma membrane. In taking into account other studies performed on this subject, as well as our own observations, we consider the material found on the surface of the chloride cells and particularly within their apical pits to be predominantly of glycoproteinous nature.

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Dédié à Monsieur le Professeur Dr Ernst Horstmann, Hambourg, à l'occasion de son soixantième anniversaire.     

The roles of serine and threonine sidechains in ion channels: a modelling study

The ion channel of the nicotinic acetylcholine receptor (nAChR) is believed to be lined by transmembrane M2 helices. A 4-8-12 sequence motif, comprising serine (S) or threonine (T) residues at positions 4, 8 and 12 of M2, is conserved between different members, anion and cation selective, of the nAChR superfamily. Parallel bundles of 4-8-12 motif-containing helices are considered as simplified models of ion channels. The relationship between S and T sidechain conformations and channelion interactions is explored via evaluation of interaction energies of K⁺ and of Cl^– ions with channel models. Energy calculations are used to determine optimal ₂ (C-C\-O-H) values in the presence of K⁺ or Cl^– ions. 4-8-12 motif-containing bundles may form favourable interactions with either cations or anions, dependent upon the ₂ values adopted. Parallel-helix and tilted-helix bundles are considered, as are heteromeric models designed to mimic the Torpedo nAChR. The main conclusion of the study is that conformational flexibility at ₂ enables both S and T residues to form favourable interactions with anions or cations. Consequently, there is apparently no difference between S and T residues in their interactions with permeant ions, which suggests that the presence of T vs. S residues within the 4-8-12 motif is not a major mechanism whereby anion/cation selectivity may be generated. The implications of these studies with respect to more elaborate models of nAChR and related receptors are considered.Abbreviations nAChR, GluR, NMDA-R, 5HT₃-R, GABA_AR, GlyR nicotinic acetylcholine, glutamate, NMDA, 5HT₃, GABA_A and glycine receptors, respectively - PhTx philanthotoxin - M2 second membrane-spanning helix of receptor-channel subunits     

In vivo phosphorylation of 2′,3′-cyclic nucleotide 3′-phosphohydrolase (CNP): CNP in brain myelin is phosphorylated by forskolin- and phorbol ester-sensitive protein kinases

2,3-cyclic nucleotide 3-phosphohydrolase (CNP) was phosphorylated in vivo, in brain slices and in a cell free system. Phosphoamino acid analysis of immunoprecipitated CNP labeled in vivo and in brain slices revealed phosphorylation of phosphoserine (94%) and phosphothreonine (5%) residues. Phosphorylation of CNP increased by 3-fold after brain slices were incubated with forskolin. Similarly, incubation of isolated myelin with [-³²]ATP with cAMP (5 M) and cAMP (5 M) + catalytic unit of cAMP dependent protein kinase dramatically increased CNP2 phosphorylation by 4- and 6-fold, respectively. It is feasible that CNP2 was predominantly phosphorylated on serine and/or threonine residues of the amino terminal peptide of CNP2, and this phosphorylation was catalyzed by protein kinase A. Phosphorylation of CNP1 and CNP2 increased 2-fold by incubating brain slices with phorbol ester. Forskolin and phorbol ester increased the phosphorylation of single, but distinct, CNP peptides. We present the first biochemical evidence that CNP2, on a protein mass basis, is far more heavily phosphorylated than CNP1, suggesting there are more phosphorylation sites on CNP2 than CNP1 and that at least one site is located on the 20-amino acid terminus of CNP2 and that is is likely a PKA site.     

Molecular environment of the phencyclidine binding site in the nicotinic acetylcholine receptor membrane

Summary Phencyclidine is a highly specific noncompetitive inhibitor of the nicotinic acetylcholine receptor. In a novel approach to study this site, a spin-labeled analogue of phencyclindine. 4-phenyl-4-(1-piperidinyl)-2.2.6.6.-tetramethylpiperidinoxyl (PPT) was synthesized. The binding of PPT inhibits⁸⁶Rb flux (IC₅₀=6.6m), and [³H] phencyclidine binding to both resting and desensitized acetylcholine receptor (IC₅₀=17 m and 0.22 m, respectively). From an indirect Hill plot of the inhibition of [³H]phencyclidine binding by PPT. a Hill coefficient of approximately one was obtained in the presence of carbamylcholine and 0.8 in -bungarotoxin-treated preparations. Taken together, these results indicate that PPt mimics phencyclidine in its ability to bind to the noncompetitive inhibitor site and is functionally active in blocking ion flux across the acetylcholine receptor channel. Analysis of the electron spin resonance signal of the bound PPT suggests that the environment surrounding the probe within the ion channel is hydrophobic, with a hydrophobicity parameter of 1.09. A dielectric constant for the binding site was estimated to be in the range of 2–3 units.     

Differences of the electrical and nicotinic receptor stimulation-evoked liberation of norepinephrine from chicken sympathetic neurons in culture: Possible involvement of different pools of the transmitter

We studied the release of [³H]norepinephrine from chicken sympathetic neurons in culture evoked by nicotinic and electrical stimulation with an intention to establish functional identity or nonidentity of the two stimuli in investigations of neurotransmitter release. Nicotinic stimulation evoked extracellular calcium dependent release of [³H]norepinephrine and the rise of intracellular calcium concentration. The release was completely blocked by nicotinic antagonists hexamethonium (100 mol/l) and mecamylamine (10 mol/l), and decreased by tetrodotoxin (0.3 mol/l) and -conotoxin (0.1 mol/l) to 17% and 27%, resp. The intracellular calcium response was decreased by nicotinic antagonists and tetrodotoxin, but not changed by -conotoxin. The electrical stimulation-evoked release was blocked by both tetrodotoxin and -conotoxin, and decreased by previous electrical, but not nicotinic, stimulation. The differential sensitivity to -conotoxin and tetrodotoxin, and the inability of nicotinic stimulation to decrease the liberation by following electrical stimulation may suggest the mobilization of different pools of the transmitter.     

Mechanisms of regulation of liver fatty acid-binding protein

Liver fatty acid-binding protein (L-FABP) expression is modulated by developmental, hormonal, dietary, and pharmacological factors. The most pronounced induction is seen after treatment with peroxisome proliferators, which induce L-FABP coordinately with microsomal cytochrome P-450 4A1 and the enzymes of peroxisomal fatty acid -oxidation. These effects of peroxisome proliferators may be mediated by a receptor which has been shown to be activated by peroxisome proliferators in mammalian cell transfection studies. However, the peroxisome proliferators tested thus far do not bind to this receptor, known as the peroxisome proliferator-activated receptor (PPAR), and its endogenous ligand(s) also remain unknown. Peroxisome proliferators inhibit mitochondrial -oxidation, and one hypothesis is that the dicarboxylic fatty acid metabolites of accumulated LCFA, formed via the P-450 4A1 -oxidation pathway, serve as primary inducers of L-FABP and peroxisomal -oxidation. We have tested this hypothesis in primary hepatocyte cultures exposed to clofibrate (CF). Inhibition of P-450 4A1 markedly diminished, via a pre-translational mechanism, the CF induction of L-FABP and peroxisomal -oxidation. In further experiments, long-chain dicarboxylic acids, the final products of the P-450 4A1 -oxidation pathway, but not LCFA, induced L-FABP and peroxisomal -oxidation pre-translationally. These results suggest a role, in part, for long-chain dicarboxylic acids in mediating the peroxisome proliferator induction of L-FABP and peroxisomal -oxidation. We also found that LCFA, which undergo rapid hepatocellular metabolism, could become inducers of L-FABP and peroxisomal -oxidation under conditions where their metabolism was inhibited. The role of the PPAR in mediating these effects is unknown, but clearly warrants further study. The induction of L-FABP and peroxisomal -oxidation by LCFA and/or their -oxidized metabolites may provide a means for limiting the deleterious effects of increased intracellular concentrations of free LCFA, and thus act as an important hepatocellular adaptation to impairment or overload of mitochondrial LCFA oxidation.     

Beta-adrenergic ([3H]CGP-12177) binding to brain slices and single intact pineal glands

We have characterized and quantified the binding of [³H]CGP-12177 to -adrenergic receptor sites in slices (300 microns) of rat cerebral cortex. The receptors are stereospecific, saturable and of high affinity. Binding of [³H]CGP is readily reversible and demonstrates appropriated drug specificty. This assay method allows the demonstration of isoproterenol-induced down-regulation (internalization) of -adrenoreceptors. Receptor recycling is observed at 37°C in the absence of -agonist but can be blocked by low temperature (0°C) or by monensin. -Adrenoreceptors can also be labeled and quantified in intact, single pineal glands of rat, mouse and hamster. Rat pineals contain approximately 10 times more binding sites than do hamster or mouse pineals and up to 8 times more sites than found in rat cerebral cortex. Rat pineal [³H]CGP binding can be up- and down-regulated but not to the same degree as seen in brain slices. This assay method is simple, rapid and provides new opportunities for the study of other receptor types in intact tissue.     

Effect of 2-fluoropalmitate,cerulenin and tunicamycin on the palmitoylation and intracellular translocation of myelin proteolipid protein

We have investigated the effect of documented protein palmitoylation inhibitors on the fatty acylation and intracellular transport of myelin proteolipid protein (PLP). To this end, brain slices from 20-day-old rats were incubated with either [³H]palmitate or [³H]leucine in the presence or absence of various concentrations of 2-fluoropalmitate (FP), cerulenin (CER), or tunicamycin (TM). FP ( 10 M) decreased the cellular uptake of [³H]palmitate and consequently reduced the labeling of palmitoyl-CoA, glycerolipids and PLP. CER ( 1 mM) reduced the palmitoylation of PLP with a concomitant decline in protein thiols. Consistent with being a fatty acyl-CoA analogue, TM ( 200 M) diminished the palmitoylation of PLP and lipids while increasing the amount of [³H]palmitoyl-CoA. Although both CER and TM decreased protein palmitoylation, only the latter affected the appearance of newly synthesized PLP into myelin. Because TM, but not CER, also reduced the formation of lipids, it is concluded that palmitoylation is not required for intracellular transport. Finally, comparison of the effect of TM in brain slices and in a cell-free system suggests that palmitoylation of PLP in whole cells may be an enzymatic process.