RESUMO
The mechanisms of peroxisomal biogenesis remain incompletely understood, specially regarding the role of the endoplasmic reticulum (ER) in human cells, where genetic disorders of peroxisome biogenesis lead to Zellweger syndrome (ZS). The Pex3p peroxisomal membrane protein (PMP) required for early steps of peroxisome biogenesis has been detected in the ER in yeast but not in mammalian cells. Here, we show that Pex3p-GFP expressed in a new ZS cell line (MR), which lacks peroxisomes due to a mutation in the PEX3 gene, localizes first in the ER and subsequently in newly formed peroxisomes. Pex3p bearing an artificial N-glycosylation site shows an electrophoretic shift indicative of ER targeting while en route to preformed peroxisomes in normal fibroblast. A signal peptide that forces its entry into the ER does not eliminate its capability to drive peroxisome biogenesis in ZS cells. Thus, Pex3p is able to drive peroxisome biogenesis from the ER and its ER pathway is not privative of ZS cells. Cross-expression experiments of Pex3p in GM623 cells lacking Pex16p or Pex16p in MR cells lacking Pex3p, showed evidence that Pex3p requires Pex16p for ER location but is dispensable for the ER location of Pex16p. These results indicate that Pex3p follows the ER-to-peroxisomal route in mammalian cells and provides new clues to understand its function.
Assuntos
Retículo Endoplasmático/metabolismo , Lipoproteínas/fisiologia , Proteínas de Membrana/fisiologia , Peroxissomos/metabolismo , Aciltransferases , Estudos de Casos e Controles , Catalase , Retículo Endoplasmático/enzimologia , Fibroblastos/citologia , Humanos , Lipoproteínas/genética , Lipoproteínas/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Mutação , Peroxinas , Transporte Proteico , Síndrome de ZellwegerRESUMO
Histamine has been proposed to be an excitatory transmitter between the carotid body (CB) chemoreceptor (glomus) cells and petrosal ganglion (PG) neurons. The histamine biosynthetic pathway, its storage and release, as well as the presence of histamine H1, H2 and H3 receptors have been found in the CB. However, there is only indirect evidence showing the presence of histamine in glomus cells, or weather its application produces chemosensory excitation. Thus, we studied the histamine immunocytochemical localization in the cat CB, and the effects of histamine, and H1, H2 and H3 receptor blockers on carotid sinus nerve (CSN) discharge, using CB and PG preparations in vitro. We found histamine immunoreactivity in dense-cored vesicles of glomus cells. Histamine induced dose-dependent increases in CSN discharge in the CB, but not in the PG. The H1-antagonist pyrilamine reduced the CB responses induced by histamine, the H2-antagonists cimetidine and ranitidine had no effect, while the H3-antagonist thioperamide enhanced histamine-induced responses. Present data suggests that histamine plays an excitatory modulatory role in the generation of cat CB chemosensory activity.
Assuntos
Corpo Carotídeo/citologia , Corpo Carotídeo/metabolismo , Células Quimiorreceptoras/metabolismo , Histamina/metabolismo , Acetilcolina/farmacologia , Potenciais de Ação/efeitos dos fármacos , Animais , Gatos , Células Quimiorreceptoras/efeitos dos fármacos , Células Quimiorreceptoras/ultraestrutura , Relação Dose-Resposta a Droga , Histamina/farmacologia , Agonistas dos Receptores Histamínicos/farmacologia , Antagonistas dos Receptores Histamínicos H1/farmacologia , Antagonistas dos Receptores Histamínicos H3/farmacologia , Hipóxia/fisiopatologia , Técnicas In Vitro , Masculino , Microscopia Imunoeletrônica/métodos , Nicotina/farmacologia , Agonistas Nicotínicos/farmacologia , Piperidinas/farmacologia , Pirilamina/farmacologia , Tirosina 3-Mono-Oxigenase/metabolismoRESUMO
The molecular pathogenesis of Alzheimer's disease (AD) involves the participation of the amyloid-beta-peptide (A beta), which plays a critical role in the neurodegeneration that triggers the disease. Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors, which are members of the nuclear receptor family. We report here that (1) PPAR gamma is present in rat hippocampal neurons in culture. (2) Activation of PPAR gamma by troglitazone and rosiglitazone protects rat hippocampal neurons against A beta-induced neurodegeneration, as shown by the 3-[4,5 -2yl]-2,5-diphenyltetrazolium bromide (MTT) reduction assay, immunofluorescence using an anti-heavy neurofilament antibody, and quantitative electron microscopy. (3) Hippocampal neurons treated with several PPAR gamma agonists, including troglitazone, rosiglitazone, and ciglitazone, prevent the excitotoxic A beta-induced rise in bulk-free Ca2+. (4) PPAR gamma activation results in the modulation of Wnt signaling components, including the inhibition of glycogen synthase kinase-3beta (GSK-3beta) and an increase of the cytoplasmic and nuclear beta-catenin levels. We conclude that the activation of PPAR gamma prevents A beta-induced neurodegeneration by a mechanism that may involve a cross talk between neuronal PPAR gamma and the Wnt signaling pathway. More important, the fact that the activation of PPAR gamma attenuated A beta-dependent neurodegeneration opens the possibility to fight AD from a new therapeutic perspective.
Assuntos
Peptídeos beta-Amiloides/toxicidade , Hipocampo/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/fisiologia , Neurônios/metabolismo , PPAR gama/metabolismo , Transdução de Sinais , Animais , Cromanos/farmacologia , Hipocampo/citologia , Humanos , Neurônios/efeitos dos fármacos , Neurônios/ultraestrutura , PPAR gama/genética , Ratos , Tiazolidinedionas/farmacologia , Troglitazona , Proteínas WntRESUMO
Hepatic ischemia-reperfusion (I-R) injury frequently is associated with cholestasis. However, the underlying mechanisms are not fully understood. The aim of the study is to assess bile secretory function in vivo in rats subjected to warm lobar hepatic ischemia at different times during reperfusion. A model of lobar 70% warm hepatic ischemia for 30 minutes was used with studies conducted at 1 and 6 hours and 1, 3, and 7 days after reperfusion. Bile secretory function was assessed after selective cannulation of bile ducts of ischemic (ILs) and nonischemic lobes (NILs). Serum activity of hepatic alanine and aspartate aminotransferase was slightly increased in rats subjected to I-R, whereas serum bile salt levels increased early during reperfusion, returning to control values after 7 days. ILs showed mild reversible leukocyte infiltration and no significant necrosis. Bile flow and bile salt excretion were significantly decreased in ILs during the first 24-hour reperfusion period compared with sham-operated rats and NILs. A marked reduction in glutathione (GSH) excretion occurred at 1 and 6 hours and 1 and 3 days, which returned to control values after 7 days. Total GSH and both reduced and oxidized GSH levels in liver homogenate and arterial blood GSH levels were unchanged at all times. Protein mass of multidrug resistance protein 2 and its function, assessed by the hepatic maximum secretory rate of ceftriaxone, did not show significant changes in ILs or NILs compared with sham-operated rats. Liver tissue gamma-glutamyl transpeptidase (GGT) and gamma-glutamylcysteine synthetase activities remained unchanged, whereas biliary GGT and cysteine secretory rates were significantly increased in ILs and NILs. Administration of acivicin, a GGT inhibitor, resulted in decreased secretion of this enzyme into bile and a parallel marked increase in biliary GSH secretion compared with untreated ischemic rats. In conclusion, warm hepatic I-R induces reversible cholestatic changes in ILs. GSH secretory rates from both ILs and NILs were markedly decreased during reperfusion. The reversibility of this effect after GGT inhibition, as well as increased release of active GGT into bile and cysteine biliary secretory rates, suggest increased GSH degradation in bile. These findings might be relevant for the I-R-induced clinical cholestasis, as well as cholangiocyte injury, seen after hepatic ischemia.
Assuntos
Bile/metabolismo , Fígado/irrigação sanguínea , Traumatismo por Reperfusão/fisiopatologia , Animais , Ácidos e Sais Biliares/sangue , Colestase/metabolismo , Colestase/fisiopatologia , Cisteína/metabolismo , Modelos Animais de Doenças , Inibidores Enzimáticos/farmacologia , Glutamato-Cisteína Ligase/metabolismo , Glutationa/sangue , Isoxazóis/farmacologia , Fígado/química , Fígado/patologia , Masculino , Proteínas Mitocondriais/fisiologia , Ratos , Ratos Sprague-Dawley , Traumatismo por Reperfusão/metabolismo , Proteínas Ribossômicas/fisiologia , Proteínas de Saccharomyces cerevisiae/fisiologiaRESUMO
Neuropeptide Y (NPY) and noradrenaline (NA) are co-transmitters at many sympathetic synapses, but it is not yet clear if their release is independently regulated. To address this question, we quantified the electrically evoked release of these co-transmitters from perivascular nerve terminals to the mesenteric circulation in control and drug-treated rats. 6-Hydroxydopamine reduced the tissue content and the electrically evoked release of ir-NPY and NA as well as the rise in perfusion pressure. A 0.001 mg/kg reserpine reduced the content of ir-NPY and NA, but did not modify their release nor altered the rise in perfusion pressure elicited by the electrical stimuli. However, 0.1mg/kg reserpine reduced both the content and release of NA but decreased only the content but not the release of ir-NPY; the rise in perfusion pressure was halved. Clonidine did not affect the release of ir-NPY while it lowered the outflow of NA, not altering the rise in perfusion pressure elicited by the electrical stimuli. Yohimbine, did not modify the release of ir-NPY but increased the NA outflow, it antagonized the clonidine effect. Therefore, presynaptic alpha2-adrenoceptors modulate the release of NA but not NPY, implying separate regulatory mechanisms.
Assuntos
Neurônios/metabolismo , Neuropeptídeo Y/metabolismo , Norepinefrina/metabolismo , Receptores Adrenérgicos alfa 2/fisiologia , Acetilcolinesterase/metabolismo , Animais , Clonidina/farmacologia , Dopamina beta-Hidroxilase/metabolismo , Relação Dose-Resposta a Droga , Imuno-Histoquímica , Microscopia de Fluorescência , Oxidopamina/metabolismo , Ratos , Ratos Sprague-Dawley , Reperfusão , Reserpina/farmacologia , Fatores de Tempo , Distribuição Tecidual , Ioimbina/farmacologiaRESUMO
We demonstrated a neutral Mg-ATPase activity in human peroxisomal membranes. To establish the precise experimental conditions for detection of this ATPase, both cytochemical and biochemical characterizations were first carried out in liver peroxisomes from control and cipofibrate-treated rats. The results demonstrated an Mg-ATPase reaction in both normal and proliferated peroxisomes. The nucleotidase activity, with marked preference for ATP, was sensitive to the inhibitors N-ethylmaleimide and 7-chloro-4-nitro-benzo-2-oxadiazole (NBDCl). An ultrastructural cytochemical analysis was developed to evaluate the peroxisomal localization, which localized the reaction product to the peroxisomal membrane. These characteristics can help to differentiate the peroxisomal ATPase from the activity found in mitochondria and endoplasmic reticulum. The conditions established for detecting the rat peroxisomal ATPase were then applied to human peroxisomes isolated from liver and skin fibroblasts in culture. A similar Mg-ATPase activity was readily shown, both cytochemically and biochemically, in the membranes of human peroxisomes. These results, together with previous evidence, strongly support the presence of a specific ATPase in the human peroxisomal membrane. This ATPase may play a crucial role in peroxisome biogenesis.
Assuntos
ATPase de Ca(2+) e Mg(2+)/análise , Histocitoquímica , Membranas Intracelulares/enzimologia , Peroxissomos/enzimologia , Trifosfato de Adenosina/metabolismo , Animais , ATPase de Ca(2+) e Mg(2+)/antagonistas & inibidores , ATPase de Ca(2+) e Mg(2+)/metabolismo , Ácido Clofíbrico/análogos & derivados , Ácido Clofíbrico/farmacologia , Retículo Endoplasmático/enzimologia , Inibidores Enzimáticos/farmacologia , Etilmaleimida/farmacologia , Ácidos Fíbricos , Fibroblastos/enzimologia , Humanos , Fígado/ultraestrutura , Mitocôndrias/enzimologia , Proliferadores de Peroxissomos/farmacologia , Ratos , Ratos Sprague-Dawley , Pele/ultraestrutura , Especificidade por SubstratoRESUMO
The relation between the expression of the oxyntic cell phenotype and the modifications of the extracellular matrix during development of the gastric glands, was studied in 10 to 21 day-old chick embryos. Cytodifferentiation of the oxyntic cells was established by ultrastructural methods, while the expression of pepsinogen, mitochondrial enzyme markers and apical secretory membranes was determined by histochemical and biochemical procedures. Results show that the morphogenesis of the glandular lobules occurs between days 8 and 15 of gestation. Later on, the lobules enlarge but maintain their basic morphology. Until day 13, the developing glands consist of primary tubes lined by a stratified columnar epithelium. The apical poles of the cells that contact the lumen show cytoplasmic processes, and Mg-ATPase activity and F-actin are concentrated at the apical cell borders. From day 13 on, the cells of the simple epithelium that lines secondary tubules budding from the primary tube, show all the features that define differentiated oxyntic cells. The synthesis of glycosaminoglycans during glandular morphogenesis was studied measuring the incorporation of radioactive sulfate into developing chick embryo proventriculi. An important increase in isotope incorporation was found between days 13 and 18 of development. Histochemical localization of these macromolecules shows that glycosaminoglycans are closely associated with the developing glandular lobules. Variations in the structure of epithelial cells undergoing morphogenesis and in the composition of the extracellular matrix are synchronous, suggesting that interactions between them may be significant in terms of the establishment and maintenance of the adult gastric gland phenotype
Assuntos
Animais , Embrião de Galinha , Matriz Extracelular , Mucosa Gástrica/embriologia , Células Parietais Gástricas/citologia , Actinas/análise , ATPase de Ca(2+) e Mg(2+)/análise , Diferenciação Celular , Complexo IV da Cadeia de Transporte de Elétrons/análise , Mucosa Gástrica/ultraestrutura , Glicosaminoglicanos/análise , MorfogêneseRESUMO
The association of myosin and a filamin-like protein to the F-actin cytoskeleton of parietal cells was studied in the rat gastric mucosa. Myosin and the filamin-like protein were localized by indirect immunofluorescence microscopy while the distribution of actin was established by using FITC-phalloidin. These cytoskeletal proteins, concentrated in the parietal cells, changed their distribution in correlation with the hydrochloric acid (HCl) secretory state of the cells and the appearance of a developed intracellular canaliculus. Thus,in resting parietal cells, actin showed a patchy distribution, delimiting the poorly developed secretory canaliculi, while myosin and the filamin-like protein distributed diffusely over the cytoplasm. In secreting cells, F-actin was concentrated in the cytoplasmic projections filling the canalicular lumen, while myosin and the filamin-like protein were excluded from this region, concentrating in the adjoining cytoplasm. The present results show that myosin and the filamin-like protein change their association with the secretory membranes in relation to the development of the secretory canaliculus of parietal cells. In resting cells, both proteins associate with the endocellular secretory membranes. In secreting cells, the microvillar projections of the canalicular surface formed by these membranes bind F-actin, but exclude myosin and the filamin-like protein