RESUMEN
The influence of the donor and the precursor of NO, namely 100 mM sodium nitroprusside and sodium nitrite on the energo-dependent Ca(2+)-transport in isolated mitochondria from rat myometrium was investigated. Changes in the mitochondrial matrix Ca(2+)-concentration was evaluated by spectrofluorimetry using Ca2+ sensitive probe Fluo-4 AM. Mg(2+)-ATP-dependent Ca(2+)-accumulation on mitochondria in the presence of succinate significantly stimulated by nitric oxide, in particular, 100 microM sodium nitroprusside amplified the transport by 1.6 times relative to its control values. NO effect becomes significant only when the incubation of mitochondria with the compounds was performed. Ca(2+)-accumulation in the presence of sodium nitroprusside effectively suppressed by protonophore (CCCP) and ruthenium red (10 microM). It was concluded that inner mitochondrial membrane Ca(2+)-uniporter stimulated by nitrogen oxide. Ca(2+)-accumulation in mitochondria in the presence of sodium nitroprusside was not sensitive to the action of a specific permeability transition pore inhibitor cyclosporine (5 microM). This data indicates that the role of permeability transition pore is less significant than Ca(2+)-uniporter in the processes of Ca(2+)-transport in mitochondria under the nitric oxide action. Thus, nitric oxide stimulates the energo-dependent Ca(2+)-accumulation by myometrium mitochondria mediated their inner membrane Ca(2+)-uniporter functioning.
Asunto(s)
ATPasa de Ca(2+) y Mg(2+)/metabolismo , Canales de Calcio/metabolismo , Calcio/metabolismo , Mitocondrias/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Membranas Mitocondriales/metabolismo , Óxido Nítrico/farmacología , Compuestos de Anilina , Animales , ATPasa de Ca(2+) y Mg(2+)/antagonistas & inhibidores , Carbonil Cianuro m-Clorofenil Hidrazona/farmacología , Ciclosporina/farmacología , Femenino , Colorantes Fluorescentes , Transporte Iónico/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Proteínas de Transporte de Membrana Mitocondrial/antagonistas & inhibidores , Membranas Mitocondriales/efectos de los fármacos , Poro de Transición de la Permeabilidad Mitocondrial , Miometrio/efectos de los fármacos , Miometrio/metabolismo , Óxido Nítrico/metabolismo , Nitroprusiato/farmacología , Ionóforos de Protónes/farmacología , Ratas , Rojo de Rutenio/farmacología , Nitrito de Sodio/farmacología , Espectrometría de Fluorescencia , Ácido Succínico/metabolismo , Ácido Succínico/farmacología , XantenosRESUMEN
Using the fluorescent probe Fluo-4 AM the authors have identified Na(+)-independent Ca2+H(+)-exchange in isolated mitochondria of rat myometrium and studied its individual properties. Formation of directional protons gradient in the matrix of mitochondria causes antyporte release of Ca2+, which has been previously accumulated in energetic processes (in the presence of Mg-ATP and succinate). The functioning of Ca2+/H(+)-exchange depends on the proton gradient and is characterized by reversibility, in case of extramitochondria environment alkalization the additional accumulation of Ca2+ by organelles is recorded. Monovalent cations gradients (Na+, K+, Li+) do not cause the release of Ca2+ from mitochondria. Rate of Ca2+/H(+)-exchange is growing in terms of increasing deltapH on the mitochondria membrane and kinetics of deltapH-induced Ca2+ release from the matrix corresponds to the laws of first order reaction. Research of Ca2+/H(+)-exchange some properties in the myometrium mitochondria showed that the above transport process is of electrogenic nature, perhaps it is done in a 1: 1 stechiometry (Hill coefficient on H+ close to 1) and is able to adjust matrix Ca2+ concentration under physiological conditions (pH activation of about 6.9). Thus, in the inner membrane of the myometrium mitochondria the available system of the secondary active Ca(2+)-transport from the matrix of these organelles to myoplasm and the functioning of Ca2+/H(+)-exchanger may underlie this process.
Asunto(s)
Antiportadores/metabolismo , ATPasa de Ca(2+) y Mg(2+)/metabolismo , Canales de Calcio/metabolismo , Calcio/metabolismo , Proteínas de Transporte de Catión/metabolismo , Mitocondrias/metabolismo , Membranas Mitocondriales/metabolismo , Miometrio/metabolismo , Adenosina Trifosfato/metabolismo , Compuestos de Anilina , Animales , Cationes Bivalentes , Cationes Monovalentes , Femenino , Concentración de Iones de Hidrógeno , Transporte Iónico , Cinética , Potencial de la Membrana Mitocondrial/fisiología , Potasio/metabolismo , Ratas , Sodio/metabolismo , Espectrometría de Fluorescencia , Ácido Succínico/metabolismo , XantenosRESUMEN
The effect of nitrosactive compounds (sodium nitroprusside and sodium nitrite) on the polarization level of the uterus myocytes inner mitochondrial membrane using the confocal laser microscopy and fluorescent probe potentialsensitive DiOC6(3) (3,3'-dihexyloxacarbocyanine) was ivestigated. Colocalisation of mitochondrial membranes specific fluorescent probes (MitoTracker Orange CM-H2TMRos, 10 - nonyl acridine orange and DiOC6(3)) was demonstrated. It was shown that sodium nitroprusside at 0.1 mM concentration caused a moderate decrease in mitochondrial transmembrane potential. That observation was confirmed by flow cytometry. Action efficiency of sodium nitrite in a similar concentration was significantly lower than that of sodium nitroprusside. It is shown that it was sodium nitroprusside which caused a slight swelling of the mitochondria. A possible protecting role of nitric oxide as to mitochondria was discussed.
Asunto(s)
Potencial de la Membrana Mitocondrial/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Membranas Mitocondriales/efectos de los fármacos , Células Musculares/efectos de los fármacos , Donantes de Óxido Nítrico/farmacología , Nitroprusiato/farmacología , Nitrito de Sodio/farmacología , Naranja de Acridina/análogos & derivados , Animales , Carbocianinas , Células Inmovilizadas , Femenino , Citometría de Flujo , Colorantes Fluorescentes , Cinética , Microscopía Fluorescente , Mitocondrias/metabolismo , Membranas Mitocondriales/metabolismo , Células Musculares/citología , Células Musculares/metabolismo , Miometrio/efectos de los fármacos , Miometrio/metabolismo , Donantes de Óxido Nítrico/química , Nitroprusiato/química , Ratas , Nitrito de Sodio/química , XantenosRESUMEN
The properties of ΔpH-induced Ca2+-transport from isolated rat myometrium mitochondria was investigated. Ca2+-accu- mulation was carried out in the presence of Mg-ATP2- and succinate. Transport of Ca2+ recorded using Ca2+-sensitive fluorescent probe Fluo-4 AM. It is shown that acidification of extramitochondrial medium is accompanied by stimulation of Ca2+ release from mitochondria. This process is insensitive to the tetraphenylphosphonium which is relatively specific Na+-Ca2+-exchanger inhibitor of mitochondrial inner membrane, but inhibited in the presence of monoclonal antibodies directed against the protein LETM1 (Anti-LETM1). LETM1 protein in some tissues is the molecular basis of the H+-Ca2+-exchanger functioning on mitochondria. It was found that the H+-Ca2+-exchanger is stimulated by 100 µM amiloride (diuretic) and inhibited by Mg ions in milimolar concentrations. The transport system was completely resistant to the action of nitric oxide (sodium nitroprusside and sodium nitrite), but was stimulated by macrocyclic compounds of Calixarenes (C-97 and C-99) in submicromolar concentrations. Thus, the mitochondria of rat myometrium probably not have a system of Na+-Ca2+-exchanger, and provide the maintenance of the matrix Ca2+-homeostasis with H+-Ca2+-exchanger. Since the transport system high affinity activated by Calixarenes, further investigation of the influence of these compounds on the transport process makes promising.
Asunto(s)
Calcio/metabolismo , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Miometrio/metabolismo , Intercambiador de Sodio-Calcio/metabolismo , Amilorida/farmacología , Compuestos de Anilina , Animales , Calixarenos/farmacología , Cationes Bivalentes , Femenino , Colorantes Fluorescentes , Concentración de Iones de Hidrógeno , Transporte Iónico/efectos de los fármacos , Magnesio/farmacología , Mitocondrias/efectos de los fármacos , Proteínas Mitocondriales/agonistas , Proteínas Mitocondriales/antagonistas & inhibidores , Miometrio/efectos de los fármacos , Óxido Nítrico/farmacología , Nitroprusiato/farmacología , Compuestos Onio/farmacología , Compuestos Organofosforados/farmacología , Protones , Ratas , Nitrito de Sodio/farmacología , Intercambiador de Sodio-Calcio/agonistas , Intercambiador de Sodio-Calcio/antagonistas & inhibidores , XantenosRESUMEN
The opportunity of Ca2+-sensitive fluorescent dye Fluo-4 AM and spectrofluorimetry method application for the study of energy-dependent Ca2+ accumulation in mitochondria from uterus smooth muscle is proved. It has been found that the presence of mitochondrial preparation increases time-dependent fluorescent response considerably and this effect depends on Ca2+ concentration in the medium. Thus, in these conditions, deesterification active probe is formed which is sensitive to Ca2+. It is shown that the accumulation of calcium ions in mitochondria in the presence of Mg-ATP and succinate depends on exogenous Ca2+ concentration and is characterized by substrate saturating. The apparent activation constant of Ca2+ accumulation is 53.9 +/- 6.9 mM, which corresponds to the physiological concentration of the cation in the cell next to mitochondria. Transit addition of Ca2+-ionophore A23187 to the incubation me- dium caused a rapid release of ionized cation from mitochondria. When proton gradient on the inner mitochondrial membrane is dissipated by protonophore CCCP, in the case of suppressing the generation of the gradient by oligomycin and in the presence of ruthenium red that inhibits Ca2+ mitochondrial accumulation systems, Ca2+ entry is significantly reduced. The results indicate the prospects of using Fluo-4 AM to study the properties of the Ca2+ accumulation system in isolated mitochondria of the myometrium.
Asunto(s)
Calcio/metabolismo , Colorantes Fluorescentes/química , Mitocondrias Musculares/metabolismo , Miocitos del Músculo Liso/metabolismo , Miometrio/metabolismo , Xantenos/química , Animales , Transporte Biológico/efectos de los fármacos , Calcimicina/farmacología , Carbonil Cianuro m-Clorofenil Hidrazona/farmacología , Fraccionamiento Celular , Femenino , Cinética , Mitocondrias Musculares/efectos de los fármacos , Miocitos del Músculo Liso/química , Miocitos del Músculo Liso/efectos de los fármacos , Miometrio/química , Miometrio/efectos de los fármacos , Oligomicinas/farmacología , Ratas , Espectrometría de Fluorescencia , Ácido Succínico/metabolismoRESUMEN
The influence of supramolecular macrocyclic compounds--calix[4]arenes C-97, C-99, C-107, which are ouabainomymetic high affinity inhibitors of Na+, K(+)-ATPase, on the polarization level of plasmic and mitochondrial membranes of rat uterine smooth muscle cells was investigated. The influence of these compounds on the myocytes characteristic size was studied. By using a confocal microscopy and specific for mitochondrial MitoTracker Orange CM-H2TMRos dye it was proved that the potential-sensitive fluorescent probe DiOC6(3) interacts with mitochondria. Artificial potential collapse of plasmic membrane in this case was modeled by myocytes preincubation with ouabain (1 mM). Further experiments performed using the method of flow cytometry with DiOC6(3) have shown that the compounds C-97, C-99 and C-107 at concentration 50-100 nM caused depolarization of the plasma membrane (at the level of 30% relative to control values) in conditions of artificial collapse of mitochondrial potential by myocytes preincubation in the presence of 5 mM of sodium azide. Under artificial sarcolemma depolarization by ouabain, calixarenes C-97, C-99 and C-107 at 100 nM concentrations caused a transient increase of mitochondrial membrane potential, that is 40% of the control level and lasted about 5 minutes. Calixarenes C-99 and C-107 caused a significant increase in fluorescence of myocytes in these conditions, which was confirmed by confocal microscopy too. It was proved by photon correlation spectroscopy method that the C-99 and C-107 caused an increase of characteristic size of myocytes.