RESUMO
Glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD) was phosphorylated in vitro by cAMP-dependent protein kinase (PKA) and by tyrosine kinase. Phosphorylation by PKA occurred in the 110 kDa native form of GPI-PLD as well as in multiple proteolytic degradation products and caused a significant decrease in enzyme activity. Dephosphorylation by treatment with alkaline phosphatase completely restored GPI-PLD activity. In addition, incubation of GPI-PLD with trypsin, which results in the generation of distinct peptide fragments, resulted in complete dephosphorylation of radiolabeled GPI-PLD. The site of phosphorylation by PKA was assigned to Thr-286. Tyrosine phosphorylation was only observed in a proteolytically processed fragment of GPI-PLD but not in the 110 kDa native form and had no effect on GPI-PLD activity.
Assuntos
Fosfolipase D/metabolismo , Animais , Bovinos , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Eletroforese em Gel de Poliacrilamida , Hidrólise , Fosfolipase D/isolamento & purificação , Fosforilação , Proteínas Tirosina Quinases/metabolismoRESUMO
Idebenone, a compound with protective efficacy against neurotoxicity both in in vitro and in in vivo models, exists in two different oxidative states: the ubiquinol-derivative (reduced idebenone) and the ubiquinone-derivative (oxidised idebenone). In the present study, we have observed that both the redox forms of idebenone have a dose-dependent inhibitory effect on the enzymatic metabolism of arachidonic acid in astroglial homogenates (IC50 reduced idebenone: 1.76 +/- 0.86 microM; IC50 oxidised idebenone: 16.65 +/- 3.48 microM), while in platelets, they are apparently less effective (IC50 reduced idebenone: 18.28 +/- 4.70 microM; IC50 oxidised idebenone: > 1 mM). We have also observed that the oxidised form preferentially inhibited cyclooxygenase vs. lipoxygenase metabolism (IC50 ratio lipoxygenase/cyclooxygenase: 3.22), while the reduced form did not discriminate between the two pathways (IC50 ratio lipoxygenase/cyclooxygenase: 1.38). In this respect, the inhibitory action of reduced idebenone resembled that of the antioxidant nordihydroguaiaretic acid, while oxidised idebenone behaved similarly as indomethacin and piroxicam--two typical anti-inflammatory agents. Our results suggest the existence of two distinct mechanisms of action for the two redox forms of idebenone and a preferential action of the drug on arachidonic acid metabolism in the central nervous system.
Assuntos
Antioxidantes/farmacologia , Ácido Araquidônico/metabolismo , Astrócitos/efeitos dos fármacos , Benzoquinonas/farmacologia , Fármacos Neuroprotetores/farmacologia , Animais , Astrócitos/metabolismo , Células Cultivadas , Cromatografia Líquida de Alta Pressão , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Masculino , Oxirredução , Prostaglandina-Endoperóxido Sintases/metabolismo , Ratos , Ratos Sprague-Dawley , Ubiquinona/análogos & derivadosRESUMO
The surface coat of procyclic forms of Trypanosoma brucei consists of related, internally repetitive glycoproteins known as EP and GPEET procyclins. Previously we showed that the extracellular domain of GPEET is phosphorylated. We now show that phosphorylation of this glycosylphosphatidylinositol-anchored surface protein can be induced in vitro using a procyclic membrane extract. Using antibodies that recognize either the phosphorylated or unphosphorylated form of GPEET, we analyzed their expression during differentiation of bloodstream forms to procyclic forms. Unphosphorylated GPEET, together with EP, was detected in cell lysates 2-4 hours after initiating differentiation whereas phosphorylated GPEET only appeared after 24 hours. Surface expression of EP and both forms of GPEET occurred after 24-48 hours and correlated with the detection of phosphorylated GPEET on immuno-blots. Electron micrographs showed that unphosphorylated GPEET was predominantly in the flagellar pocket whereas the phosphorylated form was distributed over the cell surface. In contrast, expression of a membrane-bound human placental alkaline phosphatase in procyclic forms caused the accumulation of dephosphorylated GPEET on the cell surface, while the phosphorylated form was restricted to the flagellar pocket. A GPEET-Fc fusion protein, which was retained intracellularly, was not phosphorylated. We propose that unphosphorylated GPEET procyclin is transported to a location close to or at the cell surface, most probably the flagellar pocket, where it becomes phosphorylated. To the best of our knowledge, this study represents the first localization of phosphorylated and unphosphorylated forms of a GPI-anchored protein within a cell.
Assuntos
Glicosilfosfatidilinositóis/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de Protozoários/metabolismo , Trypanosoma brucei brucei/metabolismo , Fosfatase Alcalina/genética , Animais , Transporte Biológico , Membrana Celular , Expressão Gênica , Humanos , Fragmentos Fc das Imunoglobulinas/genética , Fragmentos Fc das Imunoglobulinas/metabolismo , Glicoproteínas de Membrana/genética , Microscopia Eletrônica , Fosforilação , Proteínas de Protozoários/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Trypanosoma brucei brucei/ultraestruturaRESUMO
In many different cells, glycosylphosphatidylinositol (GPI)-anchored molecules are clustered in membrane microdomains that resist extraction by detergents at 4 degrees C. In this report, we identified the presence of such domains in human erythrocytes and examined the ability of exogenously-added GPI-anchored molecules to colocalize with the endogenous GPI-anchored proteins in these detergent-insoluble complexes. We found that the addition to human erythrocytes of three purified GPI-anchored proteins having different GPI lipid moieties resulted in their efficient and correct incorporation into the membrane. The extent of membrane insertion was dependent on the intactness of the GPI lipid moiety. However, unlike the endogenous GPI-anchored proteins, the in vitro incorporated GPI molecules were not resistant to membrane extraction by Triton X-100 at 4 degrees C. In addition, in contrast to the endogenous GPI-anchored proteins, they were not preferentially released from erythrocytes during vesiculation induced by calcium loading of the cells. These results suggest that in vitro incorporated GPI-linked molecules are excluded from pre-existing GPI-enriched membrane areas in human erythrocytes and that these microdomains may represent the sites of membrane vesicle formation.
Assuntos
Membrana Eritrocítica/metabolismo , Glicosilfosfatidilinositóis/sangue , Acetilcolinesterase/sangue , Cálcio/farmacologia , Etanolamina , Humanos , Isoflurofato , Glicoproteínas de Membrana/sangue , Proteínas de Membrana/sangue , Octoxinol/farmacologia , Fosfatidilinositol Diacilglicerol-Liase , Fosfolipase D/metabolismo , Solubilidade , Trítio , Fosfolipases Tipo C/metabolismo , Glicoproteínas Variantes de Superfície de Trypanosoma/sangueRESUMO
Long-term treatment of astrocytes in primary culture with L-glutamate (0.1-3 mM) resulted in a dose-dependent increase in D-[3H]aspartate uptake. The effect was abolished by an antagonist of kainate/AMPA receptors, CNQX, and mimicked by kainate, but not by AMPA or tACPD. Both glutamate and kainate caused a dramatic up-regulation (82% and 69%, respectively) of GLAST, a predominant glutamate transporter in cultured astroglia, though the mRNA levels appeared unaffected. Long-term treatment of cultures with dBcAMP stimulated D-[3H]aspartate uptake as well as GLAST expression. Apart from glutamate, none of the agonists used was capable of increasing further the uptake capacity of the dBcAMP-treated astroglia. The glutamate receptor-dependent modulation of glutamate transport in astroglial cultures may represent a novel feedback regulatory mechanism for glutamate uptake in the brain.
Assuntos
Astrócitos/metabolismo , Proteínas de Transporte/biossíntese , Agonistas de Aminoácidos Excitatórios/farmacologia , Glicoproteínas/biossíntese , Receptores de Glutamato/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos , Sistema X-AG de Transporte de Aminoácidos , Animais , Animais Recém-Nascidos , Ácido Aspártico/metabolismo , Astrócitos/efeitos dos fármacos , Western Blotting , Bucladesina/farmacologia , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Antagonistas de Aminoácidos Excitatórios/farmacologia , Immunoblotting , Camundongos , RNA Mensageiro/biossínteseRESUMO
In situ hybridization histochemistry for somatostatin receptors-1, -2, -3 and -4 section and receptor autoradiography using [125I]CGP 23996, [125I]somatostatin-28, [125I]seglitide and [125I]Tyr3 octreotide were carried out to determine the expression of somatostatin receptor messenger RNAs and binding sites in the hippocampus and cerebral cortex of rats 21 days following generalized limbic seizures induced by subcutaneous injection of 12mg/kg kainic acid. In control rats, somatostatin-1 to somatostatin-4 receptor messenger RNAs were found in the pyramidal layer and granule cell layer of the dentate gyrus. After kainate treatment, the CA1 subfield displayed a selective decrease in somatostatin-3 and somatostatin-4 receptor hybridization signals of 35 and 41%, respectively, whereas no changes were observed in the remaining hippocampal areas. Somatostatin-1 and somatostatin-2 receptor messenger RNA expression in the hippocampus remained unaffected by kainate treatment. No effect of kainate was observed in the expression of somatostatin receptor messenger RNAs in the cerebral cortex. In control rats, the selective somatostatin-2 receptor ligands, [125I]seglitide and [125I]Tyr3 octreotide and the non-selective somatostatin receptor ligands [125I]CGP 23996 and [125I]somatostatin-28, labelled preferentially the stratum oriens and radiatum CA1, the granule and molecular layers of the dentate gyrus and the deep layers of the cerebral cortex. [125I]somatostatin-28 and [125I]CGP 23996 labelled sites were selectively decreased by 32 and 39%, respectively, in the stratum radiatum CA1 after kainate treatment. [125I]CGP 23996 binding was also decreased by 35% in the stratum oriens CA1 and by 36% on average in the stratum oriens and radiatum CA3. [125I]seglitide and [125I]Tyr3 octreotide binding was not affected by kainate in any hippocampal region. The granule and molecular layers of the hippocampus and the layers IV-VI of the cerebral cortex did not show changes in binding sites for any of the radioligands analysed. A 18 and 35% decrease in the spontaneous and 50 mM KCl-induced somatostatin release from hippocampal slices was found two days after kainate, a likely reflection of neuronal cell loss. No differences in somatostatin release were observed 21 days after kainate treatment. At this latter time, the rats had an enhanced susceptibility to tonic-clonic seizures induced by intraperitoneal injection of 30 mg/kg pentylenetetrazol, a subconvulsant dose in naive rats. Bilateral infusion of 6 micrograms RC 160, a selective somatostatin-2 receptor agonist, in the dentate gyrus 21 days after kainate, significantly reduced (P < 0.05) the number of animals with tonic-clonic seizures induced by pentylenetetrazol.(ABSTRACT TRUNCATED AT 400 WORDS)
Assuntos
Hipocampo/metabolismo , Ácido Caínico/farmacologia , RNA Mensageiro/biossíntese , Receptores de Somatostatina/biossíntese , Somatostatina/análogos & derivados , Somatostatina/metabolismo , Sequência de Aminoácidos , Analgésicos , Animais , Autorradiografia , Sítios de Ligação/efeitos dos fármacos , Hipocampo/citologia , Hipocampo/efeitos dos fármacos , Hibridização In Situ , Masculino , Dados de Sequência Molecular , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Pentilenotetrazol/farmacologia , Ratos , Ratos Sprague-Dawley , Receptores de Somatostatina/agonistas , Receptores de Somatostatina/efeitos dos fármacos , Convulsões/induzido quimicamente , Convulsões/metabolismo , Somatostatina/farmacologiaRESUMO
While studying the enzymatic processing of arachidonic acid (AA) to eicosanoids in homogenates of hippocampal astrocytes, we observed that all the HPLC peaks corresponding to AA metabolites displayed significantly different levels depending on the presence or not of free Ca2+ in the incubation medium. A specific pattern was noticed, i.e. lipoxygenase (LOX) derivatives, in particular 12-hydroxyeicosatetraenoic acid (12-HETE), showed higher levels in medium containing 1 mM Ca2+, while cycloxygenase (COX) products including prostaglandins (PG) F2 alpha, E2 and D2 and 12-hydroxyhepatadecatrienoic acid (12-HHT), were higher in Ca(2+)-free medium. COX metabolism exceeded LOX metabolism by threefold in Ca(2+)-free medium, while it was only 60% of it in 1 mM Ca2+. The total amount of AA processed under the two conditions was identical. These data suggest that free [Ca2+] influences the pattern of AA metabolites formed in hippocampal astrocytes, with possible important implications in view of the distinct roles played by COX and LOX eicosanoids in synaptic transmission and neurotoxicity in this area.
Assuntos
Ácidos Araquidônicos/metabolismo , Astrócitos/enzimologia , Cálcio/fisiologia , Hipocampo/enzimologia , Lipoxigenase/metabolismo , Prostaglandina-Endoperóxido Sintases/metabolismo , Animais , Astrócitos/metabolismo , Células Cultivadas , Meios de Cultura , Eicosanoides/metabolismo , Hipocampo/citologia , Hipocampo/metabolismo , RatosRESUMO
We measured the release of neuropeptide Y (NPY) from hippocampal slices of rats at various times after limbic seizures induced by a subcutaneous injection of 12 mg/kg kainic acid (KA). Two days after KA, 100 mM KCl induced a 1.6 +/- 0.2-fold increase in NPY release compared to saline-injected rats (P < 0.05), while spontaneous and 50 mM KCl-induced release were unchanged. Thirty days after KA, the spontaneous and 100 mM KCl-induced efflux of NPY was enhanced 2-fold on average (P < 0.01) compared to controls, while no significant differences were found using 50 mM KCl. Tissue concentration of NPY was raised 2.2 +/- 0.2 times (P < 0.01) 30 days after KA. Thirty days after KA, the rats showed enhanced susceptibility to tonic-clonic seizures, assessed using a normally subconvulsive dose of pentylenetetrazol (PTZ; 30 mg/kg). A selective antibody (Ab) raised against NPY in a rabbit was infused bilaterally for three days in the CA3 area and dentate gyrus (DG) of the dorsal hippocampus of rats treated 30 days before with KA. This significantly reduced (P < 0.05) the number of animals with tonic-clonic seizures induced by 30 mg/kg PTZ, compared to KA treated rats which received the inactivated Ab. The Ab was ineffective in naive rats injected with a full convulsive dose of PTZ (55 mg/kg). The present results show that neuronal release of NPY is enhanced in the hippocampus after limbic seizures induced in rats by KA. This effect persists for at least 30 days and may contribute to the chronically enhanced susceptibility to seizures after injection of this toxin.
Assuntos
Hipocampo/metabolismo , Ácido Caínico , Neuropeptídeo Y/metabolismo , Convulsões/induzido quimicamente , Animais , Anticorpos/imunologia , Doença Crônica , Suscetibilidade a Doenças , Relação Dose-Resposta a Droga , Eletrofisiologia , Hipocampo/efeitos dos fármacos , Hipocampo/patologia , Técnicas In Vitro , Masculino , Neurônios/fisiologia , Neuropeptídeo Y/imunologia , Pentilenotetrazol , Cloreto de Potássio/farmacologia , Ratos , Ratos Sprague-DawleyRESUMO
Using microdialysis and a sensitive RIA, we have studied the in vivo release of the neuropeptide galanin (GAL) from the ventral hippocampus of freely moving rats. The spontaneous outflow of GAL-like immunoreactivity (GAL-LI) (1.8 +/- 0.3 fmol per ml per 20 min) was dependent on the presence of extracellular Ca2+ and was inhibited by tetrodotoxin. Evoked release induced by infusion of KCl (60 mM) or veratridine (148 microM) was also Ca(2+)-dependent and sensitive to tetrodotoxin. Electrical stimulation of the ventral limb of the diagonal band nuclei induced a frequency-dependent (50-200 Hz) and tetrodotoxin-sensitive overflow of GAL-LI in the hippocampus. In vitro GAL-LI release (1.0 +/- 0.02 fmol per ml per 5 min), studied in slices of rat ventral hippocampus, was also Ca(2+)-dependent and was increased in a concentration-dependent manner by KCl depolarization. This study demonstrates the release of the neuropeptide GAL in the rat central nervous system. The in vivo release is related to the activity of the cholinergic GAL-LI-containing cells in the septal diagonal band nuclei. The results are discussed in relation to the coexistence of GAL and acetylcholine within the septal/diagonal band complex.
Assuntos
Hipocampo/fisiologia , Neuropeptídeos/metabolismo , Peptídeos/metabolismo , Animais , Cálcio/farmacologia , Estimulação Elétrica , Feminino , Galanina , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Microdiálise , Cloreto de Potássio/farmacologia , Radioimunoensaio , Ratos , Ratos Sprague-Dawley , Tetrodotoxina/farmacologia , Fatores de Tempo , Veratridina/farmacologiaRESUMO
The mean activity of lactate dehydrogenase (LDH) in hepatocytes near the central vein region of unfixed sections of mouse liver was determined and compared with 3 different histochemical methods: conventional method, polyvinyl alcohol (PVA) method, and gel film method. An image analysis system was used for measuring the enzyme activity in single hepatocytes. The mean activities were approximately 1.4 and 2.7 times higher with the PVA and a gel film techniques respectively than with conventional aqueous media. The highest activity of LDH was obtained with gel media; this can be explained by the lowest diffusion late of this soluble cytoplasmic enzyme from the secretion into the medium. In the conventional technique, the apparent activity was found to be about 16% lower when sections were incubated vertically in a large volume of medium than when they were incubated horizontally in a small volume of medium.