Effect of Chronic Nicotine Treatment on Nicotinic Autoreceptor Function and N-[3H]Methylcarbamylcholine Binding Sites in the Rat Brain

It has been reported that N-methylcarbamylcholine (MCC), a nicotinic agonist, binds to central nicotinic receptors and causes an increase of acetylcholine (ACh) release from certain central cholinergic nerve terminals. The present experiments determine whether these two phenomena change in response to the chronic administration of nicotine, a procedure known to result in an increase in nicotinic binding sites. Chronic nicotine caused a brain region-specific up-regulation of [3H]MCC sites; binding increased in the frontal cortex, parietal cortex, striatum, and hippocampus, but not in the occipital cortex or cerebellum. The effect of nicotine was selective to nicotinic binding sites, because muscarinic sites, both M1 ([ 3H]pirenzepine) and M2 ([3H]ACh), were unaffected by chronic nicotine treatment. MCC increased the release of ACh from the frontal cortex and hippocampus by a calcium-dependent mechanism; MCC did not alter ACh release from striatum or occipital cortex of control animals. The MCC-induced increase in ACh release was not apparent in those animals which had been treated with nicotine. There was a partial recovery of nicotinic autoreceptor function when animals were allowed to recover (4 days) following chronic nicotine treatment, but the density of binding sites remained increased compared to control. Chronic nicotine did not change the potassium-evoked release of ACh from the frontal cortex or hippocampus, but decreased this measure from striatum. It also decreased the ACh content of the striatum, but not that of the cortex or the hippocampus; the activity of choline acetyltransferase was not altered in any of the regions tested.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of synaptosomes from the central nervous system of insects

Nerve terminals from the head ganglia of Locusta migratoria were isolated by means of a modified microscale flotation technique. Enzymatic, ultrastructural and chemical analysis revealed that the synaptosomal fraction was highly enriched in well-preserved nerve endings containing almost no free mitochondria. Cholinergic activities (choline acetyltransferase, acetylcholinesterase, acetylcholine receptors) were found to be concentrated in the synaptosomal fraction. The cholinergic nature and the functional integrity of nerve endings isolated from locusts were further supported by the existence of a high affinity choline uptake system, which is abolished by hemicholinium-3 as well as by low temperature, is essentially sodium dependent and inhibited by elevated potassium concentrations. After slight modifications of the gradient densities, synaptosomes could also be isolated from other insect species.     

Growth and betaine formation in Medicago sativa tissue cultures

Static and suspension tissue cultures of Medicago sativa were initiated from sterile seedlings, excised roots and aerial portions of the seedlings. Flask suspension cultures containing chlorophyll were also established. The various tissues were harvested after 30 days, growth rates were determined, and the tissue was later lyophilized. The lyophilized tissues from various batches were extracted separately with 70% EtOH. The betaine mixture was isolated from these extracts and from seeds of the plant as their tetraphenylborate salts. The betaines were separated from the latter complex via ion exchange and column chromatography and were identified by standard procedures. All of the tissues, regardless of origin, produced choline. Only chlorophyllous tissue biosynthesized stachydrine.     

Alpha-lipoic acid affects the oxidative stress in various brain structures in mice with methionine and choline deficiency

Deficiency in methionine or choline can induce oxidative stress in various organs such as liver, kidney, heart, and brain. This study was to examine the effects of alpha-lipoic acid (LA) on oxidative stress induced by methionine and choline deficiency (MCD) in several brain structures. Male mice C57BL/6 (n = 28) were divided into four groups: (1) control – continuously fed with standard chow; (2) LA – fed with standard chow and receiving LA; (3) MCD2 – fed with MCD diet for two weeks, and (4) MCD2+LA – fed with MCD diet for two weeks and receiving LA (100 mg/kg/day intraperitonealy [i.p.]). Brain tissue (cortex, hypothalamus, striatum and hippocampus) was taken for determination of oxidative stress parameters. MCD diet induced a significant increase in malondialdehyde and NOx concentration in all brain regions, while LA restored their content to normal values. Similar to this, in MCD2 group, activity of total SOD, MnSOD, and Cu/ZnSOD was reduced by MCD diet, while LA treatment improved their activities in all brain structures. Besides, in MCD2 group a decrease in catalase activity in cortex and GSH content in hypothalamus was evident, while LA treatment induced an increase in catalase activity in cortex and striatum and GSH content in hypothalamus. LA treatment can significantly reduce lipid peroxidation and nitrosative stress, caused by MCD diet, in all brain regions by restoring antioxidant enzymes activities, predominantly total SOD, MnSOD, and Cu/ZnSOD, and to a lesser extent by modulating catalase activity and GSH content. LA supplementation may be used in order to prevent brain oxidative injury induced by methionine and choline deficiency.     

Structure determinants and substrate recognition of serine carboxypeptidase-like acyltransferases from plant secondary metabolism

Structures of the serine carboxypeptidase-like enzymes 1-O-sinapoyl-beta-glucose:L-malate sinapoyltransferase (SMT) and 1-O-sinapoyl-beta-glucose:choline sinapoyltransferase (SCT) were modeled to gain insight into determinants of specificity and substrate recognition. The structures reveal the alpha/beta-hydrolase fold as scaffold for the catalytic triad Ser-His-Asp. The recombinant mutants of SMT Ser173Ala and His411Ala were inactive, whereas Asp358Ala displayed residual activity of 20%. 1-O-sinapoyl-beta-glucose recognition is mediated by a network of hydrogen bonds. The glucose moiety is recognized by a hydrogen bond network including Trp71, Asn73, Glu87 and Asp172. The conserved Asp172 at the sequence position preceding the catalytic serine meets sterical requirements for the glucose moiety. The mutant Asn73Ala with a residual activity of 13% underscores the importance of the intact hydrogen bond network. Arg322 is of key importance by hydrogen bonding of 1-O-sinapoyl-beta-glucose and L-malate. By conformational change, Arg322 transfers L-malate to a position favoring its activation by His411. Accordingly, the mutant Arg322Glu showed 1% residual activity. Glu215 and Arg219 establish hydrogen bonds with the sinapoyl moiety. The backbone amide hydrogens of Gly75 and Tyr174 were shown to form the oxyanion hole, stabilizing the transition state. SCT reveals also the catalytic triad and a hydrogen bond network for 1-O-sinapoyl-beta-glucose recognition, but Glu274, Glu447, Thr445 and Cys281 are crucial for positioning of choline.     

Localization of cholinergic innervation and neurturin receptors in adult mouse heart and expression of the neurturin gene

Neurturin (NRTN) is a neurotrophic factor required during development for normal cholinergic innervation of the heart, but whether NRTN continues to function in the adult heart is unknown. We have therefore evaluated NRTN expression in adult mouse heart and the association of NRTN receptors with intracardiac cholinergic neurons and nerve fibers. Mapping the regional distribution and density of cholinergic nerves in mouse heart was an integral part of this goal. Analysis of RNA from adult C57BL/6 mouse hearts demonstrated NRTN expression in atrial and ventricular tissue. Virtually all neurons in the cardiac parasympathetic ganglia exhibited the cholinergic phenotype, and over 90% of these cells contained both components of the NRTN receptor, Ret tyrosine kinase and GDNF family receptor α2 (GFRα2). Cholinergic nerve fibers, identified by labeling for the high affinity choline transporter, were abundant in the sinus and atrioventricular nodes, ventricular conducting system, interatrial septum, and much of the right atrium, but less abundant in the left atrium. The right ventricular myocardium contained a low density of cholinergic nerves, which were sparse in other regions of the working ventricular myocardium. Some cholinergic nerves were also associated with coronary vessels. GFRα2 was present in most cholinergic nerve fibers and in Schwann cells and their processes throughout the heart. Some cholinergic nerve fibers, such as those in the sinus node, also exhibited Ret immunoreactivity. These findings provide the first detailed mapping of cholinergic nerves in mouse heart and suggest that the neurotrophic influence of NRTN on cardiac cholinergic innervation continues in mature animals.This study was supported by the National Heart, Lung, and Blood Institute (grant HL-54633).     

Evaluation of cytochrome c affinity to anionic phospholipids by means of surface plasmon resonance

We attempted to evaluate the affinity of the anionic phospholipids to cytochrome c by means of surface plasmon resonance (SPR) technique and to correlate it with the cytochrome c active site alterations and peroxidase activity. Our experiments showed a strong interdependence between the phospholipid fatty acid saturation degree, the active site structure alterations and peroxidase activity of the cytochrome c phospholipid complex. Cytochrome c peroxidase activity and Trp59 fluorescence increase in the sequence of phosphatidyl choline (PC) → phosphatidylserine (PS) → cardiolipin (CL) → phosphatidic acid (PA). The association constant (K_a) increased in the sequence PC → PA → PS → CL. The SPR spectroscopy data shows that K_a is independent of lipid saturation degree, but correlates with phospholipid negative charge value.     

Kinesin‐2 differentially regulates the anterograde axonal transports of acetylcholinesterase and choline acetyltransferase in Drosophila

Choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) are involved in acetylcholine synthesis and degradation at pre‐ and postsynaptic compartments, respectively. Here we show that their anterograde transport in Drosophila larval ganglion is microtubule‐dependent and occurs in two different time profiles. AChE transport is constitutive while that of ChAT occurs in a brief pulse during third instar larva stage. Mutations in the kinesin‐2 motor subunit Klp64D and separate siRNA‐mediated knock‐outs of all the three kinesin‐2 subunits disrupt the ChAT and AChE transports, and these antigens accumulate in discrete nonoverlapping punctae in neuronal cell bodies and axons. Quantification analysis further showed that mutations in Klp64D could independently affect the anterograde transport of AChE even before that of ChAT. Finally, ChAT and AChE were coimmunoprecipitated with the kinesin‐2 subunits but not with each other. Altogether, these suggest that kinesin‐2 independently transports AChE and ChAT within the same axon. It also implies that cargo availability could regulate the rate and frequency of transports by kinesin motors. © 2006 Wiley Periodicals, Inc. J Neurobiol, 2006