RESUMEN
In the recent years, the toxicity of certain divalent cations has been associated with the alteration of intracellular Ca2+ homeostasis. Among other mechanisms, these cations may affect the functionality of certain Ca2+-binding proteins and/or Ca2+ pumps. The plasma membrane calcium pump (PMCA) maintains Ca2+ homeostasis in eukaryotic cells by mediating the efflux of this cation in a process coupled to ATP hydrolysis. The aim of this work was to investigate both in vitro and in cultured cells if other divalent cations (Sr2+, Ba2+, Co2+, Cd2+, Pb2+ or Be2+) could be transported by PMCA. Current results indicate that both purified and intact cell PMCA transported Sr2+ with kinetic parameters close to those of Ca2+ transport. The transport of Pb2+ and Co2+ by purified PMCA was, respectively, 50 and 75% lower than that of Ca2+, but only Co2+ was extruded by intact cells and to a very low extent. In contrast, purified PMCA-but not intact cell PMCA-transported Ba2+ at low rates and only when activated by limited proteolysis or by phosphatidylserine addition. Finally, purified PMCA did not transport Cd2+ or Be2+, although minor Be2+ transport was measured in intact cells. Moreover, Cd2+ impaired the transport of Ca2+ through various mechanisms, suggesting that PMCA may be a potential target of Cd2+-mediated toxicity. The differential capacity of PMCA to transport these divalent cations may have a key role in their detoxification, limiting their noxious effects on cell homeostasis.
Asunto(s)
Cationes/farmacocinética , Metales/farmacocinética , ATPasas Transportadoras de Calcio de la Membrana Plasmática/metabolismo , Transporte Biológico , Calcio/farmacocinética , Calmodulina/química , Calmodulina/metabolismo , Cationes/toxicidad , Células Cultivadas , Eritrocitos/citología , Eritrocitos/efectos de los fármacos , Eritrocitos/metabolismo , Células HEK293 , Humanos , Inactivación Metabólica , Metales/toxicidad , Fosfatidilserinas/metabolismo , Fosfatidilserinas/farmacología , ATPasas Transportadoras de Calcio de la Membrana Plasmática/química , Dominios ProteicosRESUMEN
The present study assessed the role of membrane components of Pseudomonas putida A (ATCC 12633) under chemical stress conditions originated by treatment with tetradecyltrimethylammonium bromide (TTAB), a cationic surfactant. We examined changes in fatty acid composition and in the fluidity of the membranes of cells exposed to TTAB at a specific point of growth as well as of cells growing with TTAB. The addition of 10-50 mg TTAB l(-1) promoted an increase in the saturated/unsaturated fatty acid ratio. By using fluorescence polarization techniques, we found that TTAB exerted a fluidizing effect on P. putida A (ATCC 12633) membranes. However, a complete reversal of induced membrane fluidification was detected after 15 min of incubation with TTAB. Consistently, the proportion of unsaturated fatty acids was lower in TTAB-treated cells as compared with non-treated cells. In the presence of TTAB, the content of phosphatidylglycerol increased (120â%), whilst that of cardiolipin decreased (60â%). Analysis of the fatty acid composition of P. putida A (ATCC 12633) showed that phosphatidylglycerol carried the major proportion of saturated fatty acids (89â%), whilst cardiolipin carried an elevated proportion of unsaturated fatty acids (18â%). The increase in phosphatidylglycerol and consequently in saturated fatty acids, together with a decrease in cardiolipin content, enabled greater membrane resistance, reversing the fluidizing effect of TTAB. Therefore, results obtained in the present study point to changes in the fatty acid profile as an adaptive response of P. putida A (ATCC 12633) cells to stress caused by a cationic surfactant.
Asunto(s)
Membrana Celular/química , Lípidos de la Membrana/análisis , Fosfolípidos/análisis , Pseudomonas putida/efectos de los fármacos , Estrés Fisiológico , Tensoactivos/metabolismo , Compuestos de Trimetilamonio/metabolismo , Cationes/metabolismo , Cationes/toxicidad , Membrana Celular/fisiología , Fluidez de la Membrana/efectos de los fármacos , Pseudomonas putida/fisiología , Tensoactivos/toxicidad , Compuestos de Trimetilamonio/toxicidadRESUMEN
The purpose of this study was to evaluate the toxicity of the oligonucleotide/cationic nanoemulsion complexes on Hep G2 cells through MTT assay. Complexes exhibit droplet size, zeta potential and viscosity of approximately 270 nm, +50mV, and 1.0 cP. Different parameters which may have an influence on toxicity results obtained by MTT assay, i.e. cells number, concentration of MTT reagent and the addition of Soerensen's glycine buffer were first evaluated. In the optimized conditions (1 x 10(4) cells and 0.5 mg/mL MTT), the overall results showed that the addition of increasing amounts of complexes (or nanoemulsions) lead to a progressive toxicity on cells attributed to the presence of the cationic lipid stearylamine in the formulations, whatever the medias's pH is. The IC50 was approximately 200 microg/ml. Such results open interesting perspectives on the use of these nanoemulsions as oligonucleotide delivery systems for Hep G2 cells.
Asunto(s)
Cationes/toxicidad , Colorantes/toxicidad , Oligonucleótidos/toxicidad , Sales de Tetrazolio/toxicidad , Tiazoles/toxicidad , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Fenómenos Químicos , Química Farmacéutica , Química Física , Ensayos de Selección de Medicamentos Antitumorales , Emulsiones , Humanos , Nanopartículas , ViscosidadRESUMEN
The capacity of Al3+-related cations (Sc3+, Ga3+, In3+, Be2+, Y3+, and La3+) to promote membrane rigidification and lateral phase separation was evaluated in liposomes containing zwitterionic (phosphatidylcholine, PC) and negatively charged (phosphatidylserine, PS) phospholipids. These effects were correlated with the capacity of the ions to stimulate Fe2+-supported lipid peroxidation. A13+, Sc3+, Ga3+, In3+, Be2+, Y3+, and La3+ (50-200 microM) increased the order parameter of the fluorescent probe 1,3-diphenylhexatriene incorporated in PC:PS membranes. In addition, the electron paramagnetic resonance spectra of spin-labeled fatty acids indicated a reduction in lipid motion induced by Sc3+, Y3+, and La3+. The effect was found to extend down to carbon 16 on the acyl chain. The ions (10-200 microM) were also able to induce lateral phase separation, as evaluated from the increase in fluorescence quenching of the probe 2-(6-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)dodecanoyl-1-hexadec anoyl-sn-glycero-3-phosphocholine. The ability of the ions to alter membrane lipid packing and induce lateral phase separation correlated in a positive manner (r2 = 0.91 and 0.90, respectively) with their capacity to stimulate the production of Fe2+-initiated 2-thiobarbituric-reactive species, a measure of lipid peroxidation. These results show that Al3+-related metal ions cause membrane rigidification and phase separation, which could affect membrane-related processes. The results support the hypothesis that ions without redox capacity can stimulate Fe2+-initiated lipid peroxidation by increasing lipid packing and by promoting the formation of rigid clusters. Both processes will bring phospholipid acyl chains closer together, thus favoring the propagation step of lipid peroxidation.