Inhibition of phosphatidylcholine and phosphatidylethanolamine biosynthesis in rat-2 fibroblasts by cell-permeable ceramides.
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Phospholipids and sphingolipids are important precursors of lipid-derived second messengers such as diacylglycerol and ceramide, which participate in several signal transduction pathways and in that way mediate the effects of various agonists. The cross-talk between glycerophospholipid and sphingolipid metabolism was investigated by examining the effects of cell-permeable ceramides on phosphatidylcholine (PtdCho) and phosphatidylethanolamine (PtdEtn) synthesis in Rat-2 fibroblasts. Addition of short-chain C6-ceramide to the cells resulted in a dose- and time-dependent inhibition of the CDP-pathways for PtdCho and PtdEtn synthesis. Treatment of cells for 4 h with 50 microM C6-ceramide caused an 83% and a 56% decrease in incorporation of radiolabelled choline and ethanolamine into PtdCho and PtdEtn, respectively. Exposure of the cells for longer time-periods (>/= 16 h) to 50 microM C6-ceramide resulted in apoptosis. The structural analogue dihydro-C6-ceramide did not affect PtdCho and PtdEtn synthesis. In pulse-chase experiments, radioactive choline and ethanolamine accumulated in CDP-choline and CDP-ethanolamine under the influence of C6-ceramide, suggesting that synthesis of both PtdCho and PtdEtn were inhibited at the final step in the CDP-pathways. Indeed, cholinephosphotransferase and ethanolaminephosphotransferase activities in membrane fractions from C6-ceramide-treated cells were reduced by 64% and 43%, respectively, when compared with control cells. No changes in diacylglycerol mass levels or synthesis of diacylglycerol from radiolabelled palmitate were observed. It was concluded that C6-ceramide affected glycerophospholipid synthesis predominantly by inhibition of the step in the CDP-pathways catalysed by cholinephosphotransferase and ethanolaminephosphotransferase. (+info)
Cationic modulation of human dopamine transporter: dopamine uptake and inhibition of uptake.
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Effects of cations on dopamine (DA) uptake into cells expressing the human dopamine transporter and on inhibition of DA uptake by various substrates and inhibitors were investigated by using rotating disk electrode voltammetry. The Na(+) dependence of DA uptake varied with Na(+) substitutes, hyperbolic with Li(+), almost linear at 1 microM DA but hyperbolic at 8 microM DA with choline, and sigmoidal with K(+). With Na(+) substituted by Li(+), K([DA]) decreased and V(app) remained constant with increasing [Na(+)], whereas K([Na+]) decreased and V(app) increased with increasing [DA], suggesting an ordered sequence with Na(+) binding before DA. Similar trends for the Na(+)-DA interactions were observed in the presence of cocaine. Cocaine inhibited DA uptake solely by increasing K([DA]), with its K(i) not significantly different at 55 and 155 mM [Na(+)], whereas it inhibited Na(+) stimulation by reducing V(app) more than K([Na+]) at 1 microM DA, and V(app) only and less potently at 8 microM DA. Thus, cocaine may compete with DA, not with Na(+), for the transporter, and might not follow a strictly ordered reaction with Na(+). With Na(+) substituted by K(+), K([DA]) or K([Na+]) became insensitive to Na(+) or DA. K(+) impaired the DA uptake mainly by reducing V(app,) but affected cocaine inhibition by elevating K(i). Despite their different patterns for inhibiting DA uptake, nontransportable inhibitors cocaine, methylphenidate, mazindol, and 1-[2-[bis(4-fluorophenyl)methoxy]ethyl]-4-(3-phenyl-2-propyl)piperazi ne (GBR12909) showed similarly modest Na(+) dependence in their K(i) values. In contrast, substrates DA, m-tyramine, and amphetamine displayed a similarly stronger Na(+) requirement for their apparent affinities. (+info)
The structural requirements for phorbol esters to enhance serotonin and acetylcholine release from rat brain cortex.
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The effects of various phorbol-based protein kinase C (PKC) activators on the electrical stimulation-induced (S-I) release of serotonin and acetylcholine was studied in rat brain cortical slices pre-incubated with [3H]-serotonin or [3H]-choline to investigate possible structure-activity relationships. 4beta-phorbol 12,13-dibutyrate (4betaPDB, 0.1-3.0 microM), enhanced S-I release of serotonin in a concentration-dependent manner whereas the structurally related inactive isomer 4alpha-phorbol 12, 13-dibutyrate (4alphaPDB) and phorbol 13-acetate (PA) were without effect. Another group of phorbol esters containing a common 13-ester substituent (phorbol 12,13-diacetate, PDA; phorbol 12-myristate 13-acetate, PMA; phorbol 12-methylaminobenzoate 13-acetate, PMBA) also enhanced S-I serotonin release with PMA being least potent. The deoxyphorbol monoesters, 12-deoxyphorbol 13-acetate (dPA), 12-deoxyphorbol 13-angelate (dPAng), 12-deoxyphorbol 13-phenylacetate (dPPhen) and 12-deoxyphorbol 13-isobutyrate (dPiB) enhanced S-I serotonin release but 12-deoxyphorbol 13-tetradecanoate (dPT) was without effect. The 20-acetate derivatives of dPPhen and dPAng were less effective in enhancing S-I serotonin release compared to the parent compounds. With acetylcholine release all phorbol esters tested had a far lesser effect when compared to their facilitatory action on serotonin release with only 4betaPDB, PDA, dPA, dPAng and dPiB having significant effects. The effects of the phorbol esters on serotonin release were not correlated with their reported in vitro affinity and isozyme selectivity for PKC. A comparison across three transmitter systems (noradrenaline, dopamine, serotonin) suggests basic similarities in the structural requirements of phorbol esters to enhance transmitter release with short chain substituted mono- and diesters of phorbol being more potent facilitators of release than the long chain esters. Some compounds notably PDA, PMBA, dPPhen, dPPhenA had different potencies across noradrenaline, dopamine and serotonin. (+info)
Antiphosphorylcholine antibody levels are elevated in humans with periodontal diseases.
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Human immunoglobulin G2 (IgG2) serum concentrations and the IgG2 antibody response to Actinobacillus actinomycetemcomitans can be influenced by genes, by environmental factors such as smoking, and by periodontal disease status. Examination of the IgG2 response to phosphorylcholine (PC), a response thought to be mainly induced by the C polysaccharide of Streptococcus pneumoniae, suggested that periodontal disease status was also associated with this response. This prompted the hypothesis that PC is an important oral antigen associated with organisms in the periodontal flora and that anti-PC antibody is elevated as a consequence of periodontal disease. Subjects in various periodontal disease diagnostic categories in which attachment loss is exhibited were tested for anti-PC in serum. Those with adult periodontitis, localized juvenile periodontitis, generalized early-onset periodontitis, and gingival recession all had similar levels of anti-PC IgG2 serum antibody which were significantly greater than in the group of subjects with no attachment loss. Analysis of plaque samples from subgingival and supragingival sites in all diseases categories for reactivity with the anti-PC specific monoclonal antibody TEPC-15 revealed that a substantial proportion of the bacteria in dental plaque (30 to 40%) bear PC antigen; this antigen was not restricted to morphotypes resembling only cocci but was also present on rods and branched filamentous organisms. We found that S. mitis, S. oralis, and S. sanguis, as well as oral actinomycetes, including A. viscosus, A. odontolyticus, and A. israelii, incorporated substantial amounts of [(3)H]choline from culture media. Further analysis of antigens derived from these organisms by Western blot indicated that S. oralis, S. sanguis, A. viscosus, A. odontolyticus, and A. israelii contained TEPC-15-reactive antigens. The data show that many commonly occurring bacterial species found in dental plaque contain PC antigen and that immunization with plaque-derived PC antigens as a consequence of inflammation and periodontal attachment loss may influence systemic anti-PC antibody concentrations. (+info)
Serum choline activates mutant acetylcholine receptors that cause slow channel congenital myasthenic syndromes.
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We have found that mutant acetylcholine receptor channels (AChRs) that cause slow-channel congenital myasthenic syndromes are activated by serum and that the high frequency of openings in serum is reduced by treatment with choline oxidase. Thus, slow-channel congenital myasthenic syndrome AChRs at the neuromuscular junction are likely to be activated both by steady exposure to serum choline and by transient exposure to synaptically released transmitter. Single-channel kinetic analyses indicate that the increased response to choline is caused by a reduced intrinsic stability of the closed channel. The results suggest that a mutation that destabilizes the inactive conformation of the AChR, together with the sustained exposure of endplates to serum choline, results in continuous channel activity that contributes to the pathophysiology of the disease. (+info)
Three-dimensional multivoxel proton MR spectroscopy of the brain in children with neurofibromatosis type 1.
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BACKGROUND AND PURPOSE: Neurofibromatosis type 1 (NF1), the most common autosomal dominant genetic disorder, frequently manifests as focal areas of signal intensity (FASI) on T2-weighted MR images. The purpose of our study was to investigate whether tumor(s), focal areas of signal intensity (FASI), and normal brain can be differentiated by using 3D multivoxel localized proton MR spectroscopy in children with neurofibromatosis type 1 (NF1) disorder. METHODS: Five children with NF1 and two healthy control subjects, all in the 3- to 11-year-old age group, were studied with a new 3D proton MR spectroscopy technique: a hybrid of 1D fourth-order transverse Hadamard spectroscopic imaging and 2D chemical shift imaging. A 3D volume-of-interest (VOI) was image-guided onto the site of the abnormality and identified on three orthogonal images. Proton MR spectroscopy partitioned the VOI into 6 x 6 x 4 (or 8 x 8 x 4) voxels, 1.5 (or 1.0) cm3 each. RESULTS: Simultaneous coverage of the entire VOI yielded good spectral signal-to-noise ratio from 136 (or 256) voxels in 27 minutes. Proton MR spectroscopy indicated that FASI a) are characterized by significantly elevated choline (Cho), reduced creatine (Cr), 2>Cho: Cr>1.3, and near normal N-acetylaspartate (NAA) levels; b) are different from tumors that exhibit Cho:Cr>2 and no NAA; c) have no intrinsic lipid or lactate signal(s); and d) correlate in spatial extent but are more extensive than indicated by MR imaging. CONCLUSION: Three-dimensional multivoxel proton MR spectroscopy reveals distinct metabolic features that differentiate normal, FASI, and tumor regions in the pediatric brain. (+info)
RNAs that bind and change the permeability of phospholipid membranes.
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The RNA world hypothesis presumes that RNA will be competent for varied essential cellular functions. One such indispensable cell function is regulation of membrane permeability. Though this was not a known RNA activity, selection-amplification yielded RNAs that bound phosphatidylcholine:cholesterol liposomes. At least eight distinct, approximately 95-mer sequences bind well to the outside of the lipid bilayer, though randomized sequences had no such activity. No distinct sequence motif for lipid binding was found. However, truncation of one such RNA shows that a smaller, 44-nucleotide irregular RNA hairpin is an active membrane binding domain. Bound RNA increases the permeability of liposomes to (22)Na(+). In addition, using voltage clamp technique, four individual RNAs increase the ion permeability of the plasma membrane of cultured human cells. The existence of multiple sequences that bind membranes and provoke permeability changes suggests that these may be elementary RNA functions that could be selected in vivo. (+info)
Recovery of dopamine neuronal transporter but lack of change of its mRNA in substantia nigra after inactivation by a new irreversible inhibitor characterized in vitro and ex vivo in the rat.
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1. In vitro, the ability of DEEP-NCS {1-[2-(diphenylmethoxy)ethyl]-4-[2-(4-isothiocyanatophenyl)ethyl]- piperazine} to inhibit [3H]-dopamine uptake by rat striatal synaptosomes was concentration-dependent and inversely related to the protein concentration. This inhibition was irreversible and resulted from changes in Vmax and KM. DEEP-NCS was less potent on noradrenaline, serotonin and choline transport. 2. One day after intrastriatal injections of DEEP-NCS (100 and 1000 pmol) in 20% dimethylsulphoxide, moderate decreases in the ex vivo dopamine uptake were observed in synaptosomes obtained from striatum injected with DEEP-NCS or solvent, and the contralateral uninjected striatum. 3. In similar conditions, 300 pmol DEEP-NCS in 45% 2 hydroxypropyl-gamma-cyclodextrin - 0.5% dimethylsulphoxide solution sub-totally reduced ex vivo dopamine uptake and mazindol binding, and moderately decreased choline and serotonin transport. These reductions were specific to DEEP-NCS-injected striata. A clomipramine pretreatment (16 mg kg-1 i.p. 1 h before) was performed in following experiments, since it reduced the DEEP-NCS-elicited decrease in serotonin uptake without affecting other indices. 4. One day after intrastriatal injection, DEEP-NCS elicited similar dose-dependent decreases in ex vivo dopamine uptake and mazindol binding (ID50=6.9-8 ng striatum-1). Changes in KM and Vmax for ex vivo dopamine transport produced by DEEP-NCS disappeared according to similar time-courses. 5. The t(1/2) for transporter recovery was 6. 1 days. This value should correspond to its actual turnover rate in vivo, since no change in transporter mRNA level was observed in substantia nigra ipsilateral to 300 pmol DEEP-NCS-injected striatum. 6. The results indicate that DEEP-NCS behaves as a potent, quite selective, irreversible inhibitor of the DAT, in vitro and in vivo. Its use in vivo suggests that the physiological half-life of the rat striatal DAT is close to 6 days. (+info)