RESUMO
Some viruses induce changes in membrane permeability during infection. We have shown previously that the porcine strain of rotavirus, OSU, induced an increase in the permeability to Na+, K+, and Ca2+ during replication in MA104 cells. In this work, we have characterized the divalent cation entry pathway by measuring intracellular Ca2+ in fura-2-loaded MA104 and HT29 cells in suspension. The permeability to Ca2+ and other cations was evaluated by the change of the intracellular concentration following an extracellular cation pulse. Rotavirus infection induced an increase in permeability to Ca2+, Ba2+, Sr2+, Mn2+, and Co2+. The rate of cation entry decreased over time as the intracellular concentration increased during the first 20 s. This indicates that regulatory mechanisms, including channel inactivation, are triggered. La3+ did not enter the cell and blocked the entry of the divalent cations in a dose-dependent manner. Metoxyverapamil (D600), a blocker of L-type voltage-gated channels, partially inhibited the entry of Ca2+ in virus-infected MA104 and HT29 cells. The results suggest that rotavirus infection of cultured cells activates a cation channel rather than nonspecific permeation through the plasma membrane. This activation involves the synthesis of viral proteins through mechanisms yet unknown. The increase in intracellular Ca2+ induced by the activation of this channel may be related to the increase in cytoplasmic and endoplasmic reticulum Ca2+ pools required for virus maturation and cell death.
Assuntos
Cálcio/metabolismo , Permeabilidade da Membrana Celular , Rotavirus/fisiologia , Animais , Células Cultivadas , Galopamil/farmacologia , Haplorrinos , Lantânio/farmacologia , Metais/metabolismoRESUMO
Rotavirus infection modifies the metabolism and ionic homeostasis of the host cell. First, there is an induction of viral synthesis with a parallel shutoff of cell protein production, followed by an increase of plasma membrane Ca2+ permeability, thereby inducing an increase of free cytoplasmic and sequestered Ca2+ concentrations. Cell death follows at a later stage. We studied the role of the increase in Ca2+ concentration in cell death. An elevation of extracellular Ca2+ concentration during infection induced an increase in [Ca2+]i and potentiated cell death. Buffering the increases in [Ca2+]i with BAPTA added at 6 h p.i. reduced the cytopathic effect without inhibiting viral protein synthesis and infectious particle production. Metoxyverapamil (D600), a Ca2+ channel inhibitor, added at 1 h p.i. reduced Ca2+ permeability, the increases in [Ca2+]i, and cell death produced by infection without modifying viral protein synthesis and infectious titer. Thapsigargin, the inhibitor of Ca(2+)-ATPase of endoplasmic reticulum, potentiated the increase of [Ca2+]i and accelerated the time course of cell death. Double staining with fluorescein diacetate and ethidium bromide or acridine orange and ethidium bromide showed that infected MA104 cells had lost plasma membrane integrity without DNA fragmentation or formation of apoptotic bodies. These results support the hypothesis that the increase in [Ca2+]i due to a product of viral protein synthesis triggers the chain of events that leads to cell death by oncosis.
Assuntos
Cálcio/metabolismo , Morte Celular , Infecções por Rotavirus/metabolismo , Animais , Bloqueadores dos Canais de Cálcio/farmacologia , ATPases Transportadoras de Cálcio/antagonistas & inibidores , Morte Celular/efeitos dos fármacos , Linhagem Celular , Permeabilidade da Membrana Celular/efeitos dos fármacos , Transformação Celular Neoplásica , Quelantes/farmacologia , Efeito Citopatogênico Viral/efeitos dos fármacos , Ácido Egtázico/análogos & derivados , Ácido Egtázico/farmacologia , Inibidores Enzimáticos/farmacologia , Galopamil/farmacologia , Haplorrinos , Homeostase , Microscopia de Fluorescência , Infecções por Rotavirus/patologia , Tapsigargina/farmacologia , Proteínas Virais/biossínteseRESUMO
Rotavirus matures inside the endoplasmic reticulum (ER), a site of intracellular calcium storage. Total cell Ca2+ depletion has been shown to impair virus maturation, arresting this process at the membrane-enveloped intermediate form following its budding into the ER. On the other hand, rotavirus infection leads to an increase in the internal Ca2+ concentration ([Ca2+]i) and sequestered Ca2+ pools. We have used thapsigargin, an inhibitor of the Ca(2+)-ATPase of the ER, to release stored Ca2+ and to study its role in rotavirus morphogenesis and cytopathic effect. Thapsigargin (0.1 to 1 microM) released stored Ca2+ from MA-104 cells, as measured by chlorotetracycline fluorescence. The concentration of cytoplasmic Ca2+, measured with fura2, increased in infected cells whether treated or not with thapsigargin. Infectivity was decreased dose dependently by thapsigargin (3 log units at 0.25 to 1 microM). In infected cells treated with thapsigargin, glycosylation of VP7 and NS28 was inhibited. Electron microscopy of infected cells treated with thapsigargin showed normal synthesis of viroplasm. However, only membrane-enveloped, not double-shelled, particles could be observed within the ER. The conformation of VP7 in infected cells treated with thapsigargin appeared to be altered, as suggested by decreased immunofluorescence reactivity with monoclonal antibodies to highly conformation-dependent VP7 epitopes. The progression of cell death in infected cells, as measured by penetration of ethidium bromide, was not affected by thapsigargin. These results indicate that rotavirus maturation depends on a high sequestered [Ca2+], specifically in the ER. Cell death is the result of the accumulation of a viral product and is not related to the production of infective particles. This viral product(s) may be responsible for the increase in [Ca2+]i, which in turn leads to cell death.
Assuntos
Cálcio/metabolismo , Rotavirus/fisiologia , Terpenos/farmacologia , Animais , Anticorpos Monoclonais/imunologia , Morte Celular/efeitos dos fármacos , Linhagem Celular , Permeabilidade da Membrana Celular/efeitos dos fármacos , Efeito Citopatogênico Viral/efeitos dos fármacos , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Homeostase/efeitos dos fármacos , Macaca mulatta , Rotavirus/efeitos dos fármacos , Rotavirus/crescimento & desenvolvimento , Rotavirus/patogenicidade , Tapsigargina , Proteínas Virais/biossíntese , Proteínas Virais/imunologia , Virulência/efeitos dos fármacosRESUMO
A fluorimetric assay using ethidium bromide (EB) was employed to quantify cell death in monolayer cell cultures (MA-104 cells) in situ and isolated cell suspensions (isolated colonic cells and Leishmania). Fluorescence of EB stained cells was measured with a photometer coupled to an inverted microscope for cell monolayers or in a spectrofluorometer for cell suspensions. Dead cells stained with trypan blue were fluorescent with EB in all preparations studied, but the latter gave an unequivocal signal. Staining with EB and fluorescein diacetate was mutually exclusive. The relationship between the number of EB fluorescent cells and the intensity of fluorescence measured in the microphotometer was linear for a large range of cell numbers (1-14000) from different types of preparations. Applicability of the method for measuring living and dead cells in two different time scales (minutes and hours) is shown using MA-104 cell monolayers infected with rotavirus and Leishmania suspensions treated with amphotericin B. The method is fast, simple, sensitive and reliable, enabling quantification of living and dead cells in monolayers and suspensions.
Assuntos
Morte Celular/fisiologia , Fluorometria , Animais , Sobrevivência Celular/fisiologia , Células Cultivadas , Chlorocebus aethiops , Colo/citologia , Etídio , Técnicas In Vitro , Leishmania braziliensis/citologia , Microscopia de Fluorescência , Ratos , Reprodutibilidade dos Testes , Infecções por Rotavirus/patologia , Sensibilidade e EspecificidadeRESUMO
The concentration and localization of serotonin was determined in the retina of the teleost Eugerres plumieri by using high performance liquid chromatography (HPLC) and immunohistochemical techniques. Serotonin and dopamine were measured simultaneously, their concentrations in the retina being 77 +/- 8 and 516 +/- 23 ng/mg tissue respectively. Treatment of the animals with pargyline significantly increased the levels of dopamine and serotonin. When retinas were treated with the neurotoxin 5,6-dihydroxytryptamine, the level of serotonin was reduced by more than 90% while the dopamine content only diminished by 20% when compared to controls. By using immunohistochemistry with a monoclonal anti-serotonin antibody it was possible to localize this amine in cell bodies of a population of amacrine cells with processes extending mainly into a thin layer of the most external lamina of the inner plexiform layer. Very few ramifications were seen projecting to the internal lamina of this layer. When visualized in flat mount preparations, dense arborization of fluorescent processes was observed. This is the first direct evidence that serotonin is apparently present in amacrine cells of the retina of E. plumieri with a distribution of the serotonergic terminals similar to goldfish but somewhat different when compared to other species.