RESUMO
Cytochrome c-mediated apoptosis in cells submitted to photodynamic therapy raises the question about the ability of photodynamically oxidized cytochrome c (cytc405) to trigger apoptosis as well as the effect of membranes on protein photo-oxidation. Cytochrome c was submitted to irradiation in the presence of MB+ in phosphate buffer and in the presence of four types of phosphatidylcholine/phosphatidylethanolamine/cardiolipin (PCPECL) liposomes (50/30/20%): totally saturated lipids (tsPCPECL), totally unsaturated lipids (tuPCPECL), partially unsaturated (80%) lipids, with unsaturation in the PC and PE content (puPCPECL80), and partially unsaturated (20%) lipids, with unsaturation in the CL content (puPCPECL20). Cytc405 was formed by irradiation in buffered water and in tsPCPECL and puPCPECL20 liposomes. In the presence of tuPCPECL and puPCPECL80, cytochrome c was protected from photodynamic damage (lipid-protected cytochrome c). In CL liposomes, 25% unsaturated lipids were enough to protect cytochrome c. The presence of unsaturated lipids, in amounts varying according to the liposome composition, are crucial to protect cytochrome c. Interesting findings corroborating the unsaturated lipids as cytochrome c protectors were obtained from the analysis of the lipid-oxidized derivatives of the samples. Native cytochrome c, lipid-protected cytochrome c, and cytc405 were microinjected in aortic smooth muscle cells. Apoptosis, characterized by nucleus blebbing and chromatin condensation, was detected in cells loaded with native and lipid protected cytochrome c but not in cells loaded with cytc405. These results suggest that photodynamic therapy-promoted apoptosis is feasible due to the protective effect of the mitochondrial lipids on the cytochrome c structure and function.
Assuntos
Grupo dos Citocromos c/química , Ácidos Graxos Insaturados/química , Lipossomos/química , Mitocôndrias Cardíacas/química , Miócitos de Músculo Liso/metabolismo , Oxigênio Singlete/química , Animais , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Células Cultivadas , Cromatina/química , Cromatina/metabolismo , Grupo dos Citocromos c/metabolismo , Grupo dos Citocromos c/farmacologia , Ácidos Graxos Insaturados/metabolismo , Ácidos Graxos Insaturados/farmacologia , Cavalos , Humanos , Peroxidação de Lipídeos/efeitos da radiação , Lipossomos/metabolismo , Lipossomos/farmacologia , Mitocôndrias Cardíacas/metabolismo , Mitocôndrias Cardíacas/efeitos da radiação , Miócitos de Músculo Liso/citologia , Oxirredução/efeitos da radiação , Fotoquimioterapia , Coelhos , Oxigênio Singlete/metabolismo , Relação Estrutura-AtividadeRESUMO
We have previously demonstrated that Chinese hamster ovary (CHO) cells transfected with the angiotensin II AT1 receptor gene containing only the coding region, presented tachyphylaxis to the total inositol phosphate (InsPs) and Ca2+ responses mediated by angiotensin II and [2-lysine]angiotensin II ([Lys2]angiotensin II). Now we have evaluated the possible role of the 3'-untranslated region of the angiotensin AT1 receptor mRNA in modulating the angiotensin AT1 receptor-mediated cellular responses. The binding parameters, as well as the Ca2+ and InsPs responses induced by angiotensin II and [Lys2]angiotensin II were similar in cells transfected with the angiotensin AT1 receptor with or without the 3'-untranslated region sequence. In cells transfected with the receptor containing the 3'-untranslated region sequence, angiotensin II-induced Ca2+ and InsPs responses were desensitized by repeated stimulations, whereas [Lys2]angiotensin II caused desensitization of InsPs production but not of Ca2+ uptake in these cells. Our results suggest that the 3'-untranslated region plays a role in modulating cell signalling involved in the tachyphylaxis of angiotensin AT1 receptor-mediated Ca2+ responses.
Assuntos
Regiões 3' não Traduzidas/fisiologia , Angiotensina II/análogos & derivados , Angiotensina II/metabolismo , Receptor Tipo 1 de Angiotensina/fisiologia , Angiotensina II/farmacologia , Animais , Sítios de Ligação , Células CHO , Cálcio/metabolismo , Cricetinae , Fosfatos de Inositol/biossíntese , Ratos , Receptor Tipo 1 de Angiotensina/genética , Receptor Tipo 1 de Angiotensina/metabolismo , Taquifilaxia/genética , Taquifilaxia/fisiologia , TransfecçãoRESUMO
The manifestation of tachyphylaxis to angiotensin II in Chinese hamster ovary (CHO) cells expressing the rat angiotensin II AT(1) receptor was investigated. The cells were transfected with a cDNA fragment containing the complete coding region of the angiotensin II AT(1A) receptor gene, as well as 56 bp of its 3'- and 52 bp of its 5'-untranslated regions. These cells (CHO-AT(1)) responded to angiotensin II by increases in intracellular Ca(2+) concentration and inositol phosphate turnover, which were inhibited upon repeated administrations, characterizing the tachyphylaxis phenomenon. In contrast to smooth muscle cells, which are rendered tachyphylactic to angiotensin II but not to [2-lysine]angiotensin II ([Lys(2)]angiotensin II), this analogue induced responses in CHO-AT(1) cells that were also inhibited upon repeated administrations. A smooth muscle cell line, which showed tachyphylaxis only to angiotensin II, became tachyphylactic also to [Lys(2)]angiotensin II after transfection with the angiotensin II AT(1) receptor gene. Our findings suggest that posttranscriptional control directed by the 3'- or the 5'-untranslated regions in the angiotensin II AT(1) receptor gene may play a role in modulating the signal transduction pathways involved in the mechanism of angiotensin II tachyphylaxis.