RESUMEN
In the study we investigated the effect of blockade cystathionine-gamma -lyase (CSE), an enzyme of hydrogen sulfide (H2S) (de novo) synthesis on the endothelium-dependent relaxation of aortic smooth muscle (SM) in old rats. It has been shown that an inhibition of CSE by propargylglycine (PAG) results in restoration of a decreased ACh-induced relaxation of aorta in old rats. This effect of PAG was removed by blocking nitric oxide (NO) synthesis in the endothelial cells. Age-related changes in the levels of H2S, NO2- and enzyme activity of the constitutive synthesis of NO (cNOS) in the heart, were determined. It has been shown that PAG introduction elevates a decreased levels of HzS, NO2- and stimulates the suppressed activity of cNOS in old rats. These results suggest that PAG activates alternative ways of H2S synthesis and stimulates the constitutive synthesis of NO. These actions of PAG restore endothelial function in old rats.
Asunto(s)
Envejecimiento/metabolismo , Alquinos/farmacología , Aorta Torácica/efectos de los fármacos , Endotelio Vascular/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Glicina/análogos & derivados , Músculo Liso Vascular/efectos de los fármacos , Vasodilatación/efectos de los fármacos , Animales , Aorta Torácica/metabolismo , Aorta Torácica/fisiopatología , Cistationina gamma-Liasa/antagonistas & inhibidores , Endotelio Vascular/metabolismo , Endotelio Vascular/fisiopatología , Glicina/farmacología , Sulfuro de Hidrógeno/metabolismo , Masculino , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/fisiopatología , Óxido Nítrico/biosíntesis , Estrés Oxidativo/fisiología , Ratas , Ratas WistarRESUMEN
Based on the fact that the acute phase of ischemic stroke is accompanied by the development of heart damage, manifestations of which are oxidative stress, morphological changes in the myocardium, in the model of brain focal ischemia-reperfusion, we investigated the oxidative stress in rat heart mitochondria and possible mechanisms of cardioprotective effect of ecdysterone. Under the conditions of brain focal ischemia-reperfusion, there is an increase rate of the generation of reactive oxygen species: superoxide (*O2-) and hydroxyl radicals (*OH), pools of stable hydrogen peroxide (H2O2), accumulate products of lipid peroxidation (diene conjugates and malonic dialdehyde), as a result of activation xanthine oxidase (marker uric acid), lipooxygenase (marker leukotriene C4) and cyclooxygenase (marker tromboksane B2) ways of *O2-(generating). In animals that received ecdysterone for 18 days, under conditions of brain focal ischemia-reperfusion, the rate of reactive oxygen species generation and the pools of lipid peroxidation products were decreased, and the survival of animals was increased. The obtained results support the development of oxidative stress in heart mitochondria of rats, powerful antiradical properties ofecdysterone, its cardioprotective effect, in conditions of brain focal ischemia-reperfusion.
Asunto(s)
Isquemia Encefálica/tratamiento farmacológico , Cardiotónicos/farmacología , Ecdisterona/farmacología , Hormonas de Invertebrados/farmacología , Mitocondrias Cardíacas/efectos de los fármacos , Daño por Reperfusión/tratamiento farmacológico , Animales , Isquemia Encefálica/metabolismo , Isquemia Encefálica/patología , Corazón/efectos de los fármacos , Peróxido de Hidrógeno/antagonistas & inhibidores , Peróxido de Hidrógeno/metabolismo , Radical Hidroxilo/antagonistas & inhibidores , Radical Hidroxilo/metabolismo , Peroxidación de Lípido/efectos de los fármacos , Lipooxigenasa/metabolismo , Malondialdehído/antagonistas & inhibidores , Malondialdehído/metabolismo , Mitocondrias Cardíacas/metabolismo , Mitocondrias Cardíacas/patología , Estrés Oxidativo , Prostaglandina-Endoperóxido Sintasas/metabolismo , Ratas , Ratas Wistar , Daño por Reperfusión/metabolismo , Daño por Reperfusión/patología , Superóxidos/antagonistas & inhibidores , Superóxidos/metabolismo , Análisis de Supervivencia , Xantina Oxidasa/antagonistas & inhibidores , Xantina Oxidasa/metabolismoRESUMEN
On the model of focal ischemia-reperfusion of the brain investigated the induction of nitrosative stress in mitochondria of rats hearts and possible mechanisms of protective action of ecdysterone. It is shown that focal ischemia-reperfusion of the brain induced in myocardial mitochondria the activation of constitutive and inducible de novo synthesis of NO by oxidation of L-arginine and not oxidative synthesis of NO through the recovery of oxidized stable metabolites of NO. Strong evidence of induction of nitrosative stress in heart mitochondria by focal ischemia-reperfusion of the brain, was a significant increase in mitochondrial pool of nitrate- and nitrite-anions and pools of nitrosothiols, that is proof of the formation and decay of peroxynitrite--a key marker of nitrosative stress. Also was observed increase in heart mitochondria by focal ischemia-reperfusion of the brain, content key regulator of de novo synthesis of NO-hydrogen sulfide and activity of inducible arginase II and, as a result, the pool of carbamide, which is also a regulator of the synthesis of NO. Previous introduction for animals herbal extract Serratsula coronata, enriched ecdysterone, reduces induction nitrosative stress in mitochondria of rats hearts under conditions of focal ischemia-reperfusion of the brain.
Asunto(s)
Antioxidantes/farmacología , Isquemia Encefálica/tratamiento farmacológico , Ecdisterona/farmacología , Mitocondrias Cardíacas/efectos de los fármacos , Extractos Vegetales/farmacología , Daño por Reperfusión/tratamiento farmacológico , Animales , Antioxidantes/química , Arginasa/genética , Arginasa/metabolismo , Arginina/metabolismo , Asteraceae/química , Isquemia Encefálica/metabolismo , Isquemia Encefálica/fisiopatología , Ecdisterona/química , Expresión Génica/efectos de los fármacos , Mitocondrias Cardíacas/metabolismo , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa de Tipo II/genética , Óxido Nítrico Sintasa de Tipo II/metabolismo , Oxidación-Reducción , Estrés Oxidativo/efectos de los fármacos , Ácido Peroxinitroso/antagonistas & inhibidores , Ácido Peroxinitroso/biosíntesis , Extractos Vegetales/química , Ratas , Ratas Wistar , Daño por Reperfusión/metabolismo , Daño por Reperfusión/fisiopatologíaRESUMEN
In experiments on isolated rat hearts, perfused according to Langendorff method, the effects of stimulation of the synthesis and blockade of endogenous hydrogen sulfide in myocardial ischemia-reperfusion (20 min/40 min) was studied. L-cysteine (121 mg/kg), precursor of endogenous hydrogen sulfide was injected intraperitoneally 30 minutes before the experiment without and within 10 minutes after administration of DL-propargylglycine (11.3 mg/kg) ("Sigma", USA)--inhibitor of cystathionine-gamma-lyase. The heart function was assessed by measuring the LVDP, dP/dt, coronary flow, heart rate. The opening of mitochondria permeability transition (MPT) pore was estimated by releasing of a stable factor with UV absorbance (lambda(max) 250 nm) into the coronary outflow probes during the initial phase of reperfusion. Administration L-cysteine was accompanied by a decrease of reperfusion disorders in cardiac function compared to control rats. The results showed that L-cysteine pretreated hearts against the blockade of cystathionine-gamma-lyase with DL-propargylglycine exerted a powerful cardioprotective effect in ischemia-reperfusion injury. Significant post-ischemic recover of heart function and improving the efficiency of oxygen metabolism was accompanied with tiny quantity of mitochondrial factor releasing comparing to I/R group. Positive influence of the combined DL-propargylglycine and L-cysteine action was the prevention of MPT pore opening.
Asunto(s)
Alquinos/farmacología , Cardiotónicos/farmacología , Cisteína/farmacología , Glicina/análogos & derivados , Corazón/efectos de los fármacos , Sulfuro de Hidrógeno/metabolismo , Daño por Reperfusión Miocárdica/prevención & control , Animales , Cardiotónicos/metabolismo , Cistationina gamma-Liasa/antagonistas & inhibidores , Cistationina gamma-Liasa/metabolismo , Cisteína/metabolismo , Glicina/farmacología , Corazón/fisiopatología , Frecuencia Cardíaca/efectos de los fármacos , Inyecciones Intraperitoneales , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/antagonistas & inhibidores , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Poro de Transición de la Permeabilidad Mitocondrial , Contracción Miocárdica/efectos de los fármacos , Daño por Reperfusión Miocárdica/metabolismo , Daño por Reperfusión Miocárdica/fisiopatología , Técnicas de Cultivo de Órganos , Ratas , Ratas WistarRESUMEN
In experiments on mitochondria isolated from the heart tissue of adult rats we studied the effects of a donor of hydrogen sulfide, NaHS, on the respiratory chain of the organelles. We found that NaHS (10(-9)-10(-6) mol/l) caused a dose-dependent decrease in the rate of oxygen consumption in the presence of succinate and ADP (state 3 to Chance), and in the absence of ADP (state 4). The decrease in the rate of oxygen consumption in a concentration NaHS 10(-9) mol/l and 10(-8) mol/l associated with an increased conjugation of oxidation and phosphorylation, as evidenced by the increase in the respiratory control, the efficiency of oxidative phosphorylation (ADP/O) is not changed. Our studies suggest a protective effect of hydrogen sulfide donor on the functional state of the mitochondria. To elucidate of other the mechanisms of the protective action H2S we also investigated the effect of hydrogen sulfide donor on the mitochondrial swelling. It was found that NaHS in the range of concentration 10(-12) - 10(-4) mol/l influences the level of mitochondria swelling of the rats heart in the dose-dependent manner. It was also shown that when the concentration of Ca2+ 1 nmol/mg protein in the medium, under the action of hydrogen sulfide in the donor concentration range 10(-12) - 10(-8) mol/l, there was a moderate swelling of rats heart mitochondria. Under the action of NaHS at a concentration of 10(-9) mol/l it was observed swelling of the mitochondria, the maximum change in the level of which was 11%. Inhibitor of mitochondrial ATP-sensitive K+ channels (K(ATP) channels) 5-hydroxydecanoate (10(-4) mol/l) partially reduced the mitochondrial swelling in the presence of NaHS (10(-9) mol/l), which may indicate the activation of K(ATP) channels. Our studies point for possible involvement of mitochondrial K(ATP) channels in implementation of the mechanisms of H2S.
Asunto(s)
Sulfuro de Hidrógeno/farmacología , Mitocondrias Cardíacas/efectos de los fármacos , Mitocondrias Cardíacas/fisiología , Adenosina Difosfato/farmacología , Animales , Relación Dosis-Respuesta a Droga , Transporte de Electrón/efectos de los fármacos , Técnicas In Vitro , Mitocondrias Cardíacas/metabolismo , Dilatación Mitocondrial/efectos de los fármacos , Fosforilación Oxidativa/efectos de los fármacos , Bloqueadores de los Canales de Potasio/farmacología , Canales de Potasio/metabolismo , Ratas , Ratas Wistar , Succinatos/farmacología , Sulfuros/farmacologíaRESUMEN
In experiments in vivo and in vitro on the mitochondria isolated from the control and spontaneously hypertensive rats (SHR) hearts, we studied the effects of a donor of hydrogen sulfide (H2S), NaHS, and H2S biosynthesis substrate, L-cysteine, on the sensitivity of the mitochondrial permeability transition pore (mPTP) opening to its natural inductor, Ca2+. We found that NaHS (10(-4), 10(-5) and 5 10(-5) mol/l) influenced the mitochondrial swelling in a concentration-dependent manner in control and spontaneously hypertensive rats. The H2S donor NaHS used in physiological concentrations (10(-6), 10(-5) and 5 10(-5) mol/l) exerted the inhibiting effect on the Ca(2+)-induced mPTP opening in control hearts (corresponding values of such effect were 31, 76, and 100%, respectively), while in spontaneously hypertensive rats hearts the protector effect of NaHS was observed only at its concentration of 10(-5) - 10(-4) mol/l. In experiments in vivo, single intraperitoneal injections of L-cysteine (10(-3) mol/kg) resulted in a decrease in the sensitivity of mPTP to it's inductor Ca2+ in control rats and SHR. In experiments in vivo in which we used a specific blocker of cystathionine-gamma-lyase, propargylglycine (10(-4) mol/kg), with the further injections of L-cysteine we observed a decrease in the threshold Ca2+ concentration (that induce the mitochondrial swelling) by three orders of magnitude in SHR, but in control rats did not effect of L-cysteine. Thus, both endogenous and exogenous hydrogen sulfide inhibits Ca(2+)-induced mitochondrial permeability transition pore opening, indicating its protective effect on pore formation in spontaneously hypertensive rats hearts. Therefore, our studies are indicative of the involvement of H2S in modulation of changes in the permeability of mitochondrial membranes, which can be an important regulatory factor in the development of cardiovascular diseases.
Asunto(s)
Calcio/farmacología , Sulfuro de Hidrógeno/farmacología , Mitocondrias Cardíacas/efectos de los fármacos , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Membranas Mitocondriales/efectos de los fármacos , Alquinos/farmacología , Animales , Calcio/metabolismo , Cistationina gamma-Liasa/antagonistas & inhibidores , Cistationina gamma-Liasa/metabolismo , Cisteína/metabolismo , Cisteína/farmacología , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Glicina/análogos & derivados , Glicina/farmacología , Inyecciones Intraperitoneales , Mitocondrias Cardíacas/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/agonistas , Proteínas de Transporte de Membrana Mitocondrial/antagonistas & inhibidores , Membranas Mitocondriales/metabolismo , Poro de Transición de la Permeabilidad Mitocondrial , Dilatación Mitocondrial/efectos de los fármacos , Ratas , Ratas Endogámicas SHR , Ratas Wistar , Sulfuros/metabolismo , Sulfuros/farmacologíaRESUMEN
In experiments in vitro on the mitochondria isolated from the rat's heart we studied the effects of the openers of ATP-sensitive potassium channels (K(ATP)-channels), flocalin and tioflocalin, on the calcium-induced mitochondrial pore (MPTP) opening. Flocalin and tioflocalin caused moderate Ca(2+)-independent mitochondria swelling, which was prevented by a specific inhibitor of 5-hydroxydecanoate. This allowed to identify these compounds as mitochondrial K(ATP)-channels openers. We found that concentration-dependent inhibitory effects (10(-7) to 10(-4) M) of flocalin (with IC50 = 50 microM) and tioflocalin (with IC50 = 2,7 microM) on Ca(2+)-induced mitochondrial swelling (MPTP opening) in the heart characterized more powerful cardioprotective action of the latter. It was shown that the administration of these compounds in experiments in vivo decreased the sensitivity of the MPTP opening to Ca2+. Thus, under physiological conditions the activators K(ATP)-channels probably provide the membrane-stabilizing effects, thereby effectively increasing the organelles resistance to Ca2+, an inductor of MPTP. The results obtained allowed to characterize the role of the compound studied as cardioprotectors and regulators of the MPTP formation in the heart, indicated their anti-ischemic and anti-apoptotic effects that can be used in order to correct the mitochondrial dysfunction under pathological conditions of the cardiovascular system.
Asunto(s)
Calcio/farmacología , Cardiotónicos/farmacología , Activación del Canal Iónico/efectos de los fármacos , Mitocondrias Cardíacas/efectos de los fármacos , Pinacidilo/análogos & derivados , Canales de Potasio/metabolismo , Animales , Cardiotónicos/química , Relación Dosis-Respuesta a Droga , Técnicas In Vitro , Mitocondrias Cardíacas/metabolismo , Dilatación Mitocondrial/efectos de los fármacos , Pinacidilo/química , Pinacidilo/farmacología , Ratas , Ratas WistarRESUMEN
In experiments vitro on the mitochondria isolated from adult and spontaneous hypertensive rat hearts, we studied the sensitivity of the mitochondrial permeability transition pore (mPTP) opening to its natural inductor, Ca2+. We observed an increase in the sensitivity of mPTP opening to Ca2+ in the heart of spontaneous hypertensive rats because of a decrease in the threshold concentration of this ion required for organelles swelling by two orders of magnitude. It was shown that the classical inhibitor mPTP cyclosporin A (10(-5) mol/L) partially (54%) inhibited of mPTP opening in the heart of these animals, indicating that the presence in the heart of these animals of cyclosporin A-insensitive component of the mPTP. The results of our observations give reason to conclude that the hypertensive state of the organism is associated with mitochondrial dysfunction, which is characterized, in particular, by an increased sensitivity mPTP to Ca2+, eliciting a widespread tissue damage and diseases of the cardiovascular system, especially hypertension.
Asunto(s)
Calcio/metabolismo , Ciclosporina/farmacología , Inhibidores Enzimáticos/farmacología , Mitocondrias Cardíacas/efectos de los fármacos , Proteínas de Transporte de Membrana Mitocondrial/antagonistas & inhibidores , Animales , Señalización del Calcio/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Mitocondrias Cardíacas/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Poro de Transición de la Permeabilidad Mitocondrial , Dilatación Mitocondrial/efectos de los fármacos , Ratas , Ratas Endogámicas SHR , Ratas WistarRESUMEN
The study was conducted on three groups of rats: Group I included Wistar rats with normal blood pressure (first control group); group II - rats with genetically determined hypertension (second control group); group Ill - rats with genetically determined hypertension under the influence ofmagnetic-laser power (study group). For the low-intensively magnetic-laser influence (MLI) we have used device MIT-MT, Ukraine, which was designed for the treatment of low-frequency magnetic field using optical flow blue and red ranges of spectrum. The MLI duration was 15 minutes for the blue range, and 25 minutes for the red one. Biochemical studies included the determination of the activity of isoenzymes of NO-synthase: constitutive (cNOS) and inducible (iNOS), the content of free hemoglobin, stable metabolites of NO, namely nitrite - (NO2(-)) and nitrate - (NO3(-)) anions, resistance to acid hemolysis of red blood cells. The contractile activity of smooth muscles of the aorta was measured. We found that magnetic-laser exposure of rats with genetically determined hypertension in the red (630 nm) and blue (470 nm wavelength) optical range even after a single session leads to an increased synthesis of nitric oxide in the blood plasma. Our data sindicate that the most effective in the intensification of endogenous nitric oxide (increase of NO2(-) and reduction of NO3(-)) and endothelium-dependent responses of aorta in rats with genetically determined hypertension was a ten-day course of the magnetic-laser exposure in the optical flow of the blue spectral range. Also, after 10 sessions of magnetic-laser exposure in rats from the above specified spectrum a stabilization of erythrocyte membranes was observed.
Asunto(s)
Aorta/efectos de la radiación , Hipertensión/radioterapia , Terapia por Luz de Baja Intensidad , Magnetoterapia , Contracción Muscular/efectos de la radiación , Músculo Liso Vascular/efectos de la radiación , Óxido Nítrico/sangre , Acetilcolina/farmacología , Animales , Aorta/efectos de los fármacos , Aorta/metabolismo , Aorta/fisiopatología , Eritrocitos/efectos de la radiación , Hemoglobinas/metabolismo , Hemólisis/efectos de la radiación , Hipertensión/sangre , Hipertensión/fisiopatología , Rayos Láser , Contracción Muscular/efectos de los fármacos , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/fisiopatología , Nitratos/metabolismo , Óxido Nítrico Sintasa de Tipo II/metabolismo , Óxido Nítrico Sintasa de Tipo III/metabolismo , Nitritos/metabolismo , Nitroprusiato/farmacología , Técnicas de Cultivo de Órganos , Ratas , Ratas Endogámicas SHR , Ratas WistarRESUMEN
The present study was aimed to investigate the effect of H2S donor (NaHS) on heart function in conditions of functional loads and ischemia-reperfusion (I/R) injury by using Langendorf isolated heart perfusion. NaHS ("Sigma", 7,4 mg per kg) was dissolved in physiological solution and injected intraperitoneally 30 min before experiment. Rat isolated hearts were Langendorf-perfused and subjected to 20-minute non-flow ischemia followed by 40-minute reperfusion. The heart function was assessed by measuring the LVDP, dP/dt, coronary flow, heart rate. The opening of mitochondria permeability transition (MPT) pore was estimated by releasing of a stable factor with UV absorbance (lambdamax 250 nm) into the coronary outflow probes during the initial phase of reperfusion. The results showed that NaHS pretreated hearts developed greater LVDP without decreasing ofdP/dt min in response to an increase of left ventricle volume indicating greater functional reserves and effectiveness of Frank-Starling low realization. NaHS increased cardiac mitochondrial membrane potential but did not changed UCP3 gene expression. Significant post-ischemic recover of heart function in NaHS group was accompanied with tiny quantity ofmitochondrial factor releasing comparing to I/R group (p<0.001). Thus, NaHS do provides cardioprotective effect by inhibition of MPT pore opening.
Asunto(s)
Cardiotónicos/farmacología , Corazón/efectos de los fármacos , Sulfuro de Hidrógeno/farmacología , Proteínas de Transporte de Membrana Mitocondrial/antagonistas & inhibidores , Daño por Reperfusión Miocárdica/tratamiento farmacológico , Sulfuros/metabolismo , Animales , Expresión Génica/efectos de los fármacos , Corazón/fisiopatología , Frecuencia Cardíaca/efectos de los fármacos , Inyecciones Intraperitoneales , Canales Iónicos/genética , Canales Iónicos/metabolismo , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Mitocondrias Cardíacas/efectos de los fármacos , Mitocondrias Cardíacas/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Poro de Transición de la Permeabilidad Mitocondrial , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Contracción Miocárdica/efectos de los fármacos , Daño por Reperfusión Miocárdica/metabolismo , Daño por Reperfusión Miocárdica/fisiopatología , Técnicas de Cultivo de Órganos , Ratas , Ratas Wistar , Proteína Desacopladora 3RESUMEN
The effect of endogenous and exogenous hydrogen sulfide (H2S) on contractile activity of vascular smooth muscle (VSM) was studied. The introduction of substrate synthesis H2S L-cysteine and its donor NaHS in vitro caused concentration-dependent relaxation of VSM of aorta and portal vein. Low concentrations of hydrogen sulfide donor (10(-5) mol/L) caused vasoconstriction of both types of the vessels. It was shown that the reaction of relaxation of VSM in response to NaHS is independent from endothelium. It was revealed that VSM of portal vein are more sensitive to the effects of H2S than VSM of aorta. Removing of aorta periadventitial adipose tissue showed no relaxation reply to the hydrogen sulfide donor NaHS in 70% of experiments. Some of the cellular mechanisms of hydrogen sulfide action were established, namely relaxation of aorta is depended on K(ATP) channel activation. This is manifested by a lack of relaxation of the aortic VSM due to K(ATP) channel inhibitor glibenclamide.
Asunto(s)
Aorta Torácica/efectos de los fármacos , Sulfuro de Hidrógeno/farmacología , Contracción Muscular/efectos de los fármacos , Músculo Liso Vascular/efectos de los fármacos , Vena Porta/efectos de los fármacos , Animales , Aorta Torácica/metabolismo , Aorta Torácica/fisiología , Cisteína/farmacología , Relación Dosis-Respuesta a Droga , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/metabolismo , Endotelio Vascular/fisiología , Gliburida/farmacología , Técnicas In Vitro , Canales KATP/metabolismo , Contracción Muscular/fisiología , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/fisiología , Vena Porta/metabolismo , Vena Porta/fisiología , Ratas , Ratas Wistar , Sulfuros/farmacología , Vasoconstricción/efectos de los fármacos , Vasoconstricción/fisiologíaRESUMEN
The ischemic preconditioning (IPC) limits myocardial injury provoked by a subsequent prolonged ischemia-reperfusion (I/ R). The underlying mechanisms of enhanced resistance of heart are actively studied, but for sure it was established that mitochondria play a major role in IPC-stimulated adaptation to ischemia. In this article we present and discuss evidences that cardioprotective effect of IPC is mediated by inhibition of mitochondria permeability transition pore (MPTP) opening. It was shown that IPC effectively prevents the excessive production of ROS by mitochondria during I/R and promotes a more complete restoration of function of isolated rat hearts. It was revealed that MPTP formation due to I/R was inhibited in IPC heart. Mitochondrial factor, the marker of MPTP opening found in outflow probes, was released in much lesser amounts in IPC hearts that in non-IPC. Furthermore, mitochondria isolated from IPC hearts showed a decreased sensitivity to calcium ions, a MPTP inductor, and, thus, massive MPTP-depended swelling of mitochondria was abrogated in IPC hearts. In our experiments we observed slight increase in inducible NOS activity right after short ischemic stimuli. We suppose that increased NO production by iNOS is involved in inhibition of MPTP and this may be one of the possible mechanisms of decreased sensitivity of mitochondria to calcium ions. It is concluded that among the processes involved in formation of cardioprotective effect of IPC, a reduction of membrane permeability due to the inhibition of MPTP opening plays a crucial role.
Asunto(s)
Precondicionamiento Isquémico Miocárdico , Mitocondrias Cardíacas/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Daño por Reperfusión Miocárdica/metabolismo , Óxido Nítrico/biosíntesis , Animales , Técnicas In Vitro , Masculino , Malondialdehído/metabolismo , Mitocondrias Cardíacas/enzimología , Poro de Transición de la Permeabilidad Mitocondrial , Daño por Reperfusión Miocárdica/enzimología , Daño por Reperfusión Miocárdica/prevención & control , Óxido Nítrico Sintasa de Tipo II/metabolismo , Estrés Oxidativo , Permeabilidad , Ratas , Ratas Wistar , Especies Reactivas de Oxígeno/metabolismoRESUMEN
We carried out a study on elderly people (70 +/- 2,2 years old) operated on as an acute occlusion of the femoral artery at its different anatomical levels. In those people, development of oxidative stress has been shown in injured lower extremity in the process of restoring of circulation. Marked increase in catalase activity in the blood gives evidence for work of the anti-oxidative defence system with tense. We determined an increase in aspartataminotransferase, alaninaminotransferase and creatininkinase in ischemia/reperfusion which are widely used to identify injures in muscular tissues. It has been shown that NO(2-) content in the blood at reperfusion depended on the level of nitrites and the duration ofischemia. Significant changes in NO metabolite content in the blood of the patients suffering from acute occlusion injures of the arteries in the lower extremities could be related with endothelial dysfunction.
Asunto(s)
Antioxidantes/metabolismo , Endotelio Vascular/metabolismo , Extremidad Inferior/irrigación sanguínea , Estrés Oxidativo , Daño por Reperfusión/metabolismo , Enfermedad Aguda , Anciano , Alanina Transaminasa/sangre , Alanina Transaminasa/metabolismo , Arteriopatías Oclusivas/complicaciones , Arteriopatías Oclusivas/cirugía , Aspartato Aminotransferasas/sangre , Aspartato Aminotransferasas/metabolismo , Catalasa/sangre , Catalasa/metabolismo , Creatina Quinasa/sangre , Creatina Quinasa/metabolismo , Endotelio Vascular/enzimología , Femenino , Arteria Femoral/enzimología , Arteria Femoral/metabolismo , Arteria Femoral/cirugía , Humanos , Masculino , Nitritos/sangre , Daño por Reperfusión/enzimología , Daño por Reperfusión/etiologíaRESUMEN
We investigated the mitochondrial membrane potential and processes of respiration and oxidative phosphorylation in suspension of cardiac mitochondria from 6 month old spontaneously hypertensive rats (SHR) and Wistar (as a control) male rats. The mitochondrial membrane potential and the speed of oxygen consumption were measured using the method described by M.Brand (1995). Processes of respiration and oxidative phosphorylation in cardiac mitochondria were measured using Oxygraph (Hansatech instruments, Norfolk, England). It has been found that in SHR the mitochondrial membrane potential was lower (-113.76 mV+/-3.65 mV) compared to Wistar rats (DeltaPsim=-152.85 mV+/-13.52 mV, p<0.01). In SHR, the respiration rate in state V2 by Chance and V3 were increased compared to Wistar rats (p<0.001). The respiration control by Chance was depressed by 23.9% in SHR compared to Wistar rats. Our data demonstrate, that SHR have some features of functioning of cardiac mitochondria which distinguish them from the Wistar rats. Our data suggest a functional link between mitochondrial energy supply and arterial hypertension.
Asunto(s)
Hipertensión/fisiopatología , Potencial de la Membrana Mitocondrial/fisiología , Mitocondrias Cardíacas/fisiología , Consumo de Oxígeno/fisiología , Animales , Metabolismo Energético , Hipertensión/genética , Hipertensión/metabolismo , Masculino , Mitocondrias Cardíacas/metabolismo , Fosforilación Oxidativa , Ratas , Ratas Endogámicas SHR , Ratas WistarRESUMEN
We explored the effect of inhibitor of mitochondrial Na(+)-Ca(2+)-exchanger CGP37157 and the effect of mitochondria permeability transition pore inhibitor, cyclosporine A, on the membrane potential and electrical responses to endothelium-dependent dilators in intact endothelial cells from excised rat aorta and EA.hy 926 endothelial cells. Cyclosporin A did not affect the resting membrane potential and hyperpolarization to acetylcholine and histamine in intact and cultured cells. In contrast, CGP37157 (20 mcM) evoked membrane depolarization in unstimulated cells and suppressed the sustained component of hyperpolarization to acetylcholine and histamine both in intact and cultured endothelial cells, respectively. This was accompanied by a pronounced depolarization with membrane potential oscillations, which were not observed in the absence of Ca2+. We conclude that CGP37157 modulates endothelial membrane potential at rest and electrical responses to endothelium-dependent dilators. Possible mechanisms accompanying the CGP37157 action are discussed.
Asunto(s)
Polaridad Celular/efectos de los fármacos , Clonazepam/análogos & derivados , Células Endoteliales/efectos de los fármacos , Potenciales de la Membrana/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Intercambiador de Sodio-Calcio/antagonistas & inhibidores , Tiazepinas/farmacología , Animales , Aorta Torácica/citología , Aorta Torácica/efectos de los fármacos , Aorta Torácica/metabolismo , Técnicas de Cultivo de Célula , Células Cultivadas , Clonazepam/farmacología , Células Endoteliales/metabolismo , Células Endoteliales/fisiología , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/metabolismo , Histamina/farmacología , Mitocondrias/metabolismo , Técnicas de Placa-Clamp , RatasRESUMEN
In experiments in vivo and in vitro on the mitochondria isolated from adult and old rat hearts, we studied the effects of a donor of hydrogen sulfide (H2S), NaHS, and H2S biosynthesis substrate, L-cysteine, on the sensitivity of the mitochondrial permeability transition pore (mPTP) opening to its natural inductor, Ca(2+). We found that NaHS (10(-12) to 10(-4) mol/l) influenced mitochondrial swelling in a concentration-dependent manner. It was also demonstrated that the addition of NaHS (10(-12) to 10(-8) mol/l) to the calcium-free medium resulted in moderate a swelling of mitochondria from both adult and old rat hearts. At 10(-10) mol/l NaHS, the maximal values of the mitochondrial swelling observed in both adult and old hearts were 11 and 15 ,%, respectively. A specific inhibitor of KATP channels, 5-hydroxydecanoate (5-HD; 10(-4) mol/l) decreased the mitochondrial swelling in the presence of NaHS (10)-10) mol/l), which can be indicative of the contribution of these channels to the calcium-independent conductance of the mitochondrial membranes in the rat hearts. The H2S donor NaHS used in physiological concentrations (10(-6), 10(-5) and 5 10(-5) mol/l) exerted the inhibiting effect on the Ca(2+)-induced mPTP opening in adult hearts (corresponding values of such effect were 31, 76, and 77%, respectively), while in old hearts the protector effect of NaHS was observed only at its concentration of 10(-5) mol/l. Therefore, the donor of H2S used in the tested concentrations (10(-12) to 10(-4) mol/l) exerted ambiguous effect on the mitochondrial swelling: low concentrations of NaHS (10(-12) to 10(-8) mol/l) increased the mitochondrial swelling, while its physiological concentrations (10(-6) to 5 10(-5) mol/l) exerted the protective effect on Ca(2+)-induced mitochondrial swelling in adult and old hearts. Pre-incubation of isolated mitochondria with 5-HD (10(-4) mol/l) resulted in a decrease in the protective effect evoked by NaHS (10(-5) mol/l) on Ca(2+)-induced mPTP opening, which is indicative of the possible involvement of mitochondrial KATP-channels in the H2S-dependent inhibition of mPTP formation in both adult and old rat hearts. In experiments in vivo, single intraperitoneal injections of both NaHS (10(4) mol/kg) and L-cysteine ((10(-3) mol/kg) resulted in a decrease in the sensitivity of mPTP to its inductor Ca(2+) in adult and old rat hearts. The action of L-cysteine, as compared with that of NaHS, was more effective in prevention of Ca(2+)-induced mitochondrial swelling. We observed a rise in Ca(2+) concentration by one order of magnitude, which evoked the mitochondrial swelling in adult and old hearts. In experiments in vivo in which we used a specific blocker ofcystathionine-g-lyase, propargylglycine (10(-4) mol/kg) that is involved in the synthesis of H2S, we observed an increase in the sensitivity of mPTP opening in old hearts because of a decrease in the threshold Ca(2+) concentration required for mitochondrial swelling by two orders of magnitude. We demonstrate the involvement of endogenous H2S in the control of mPTP formation in adult and old hearts. Our studies are indicative of the involvement of H2S in modulation of changes in the permeability of mitochondrial membranes, which can be an important regulatory factor in the development of cardiovascular diseases.
Asunto(s)
Envejecimiento/metabolismo , Calcio/farmacología , Sulfuro de Hidrógeno/farmacología , Mitocondrias Cardíacas/efectos de los fármacos , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Animales , Ácidos Decanoicos/farmacología , Relación Dosis-Respuesta a Droga , Hidroxiácidos/farmacología , Técnicas In Vitro , Membranas Intracelulares/efectos de los fármacos , Membranas Intracelulares/metabolismo , Mitocondrias Cardíacas/metabolismo , Poro de Transición de la Permeabilidad Mitocondrial , Dilatación Mitocondrial/efectos de los fármacos , Bloqueadores de los Canales de Potasio/farmacología , Ratas , Ratas Wistar , Sulfuros/farmacologíaRESUMEN
The functional significance of uncoupling proteins UCP2 and UCP3 in the cell and the organism remains unknown. There have been reports about their involvement in cellular protection mechanisms underlying the phenomenon of ischemic preconditioning. The purpose of this study was to elucidate the role of uncoupling proteins UCP2 and UCP3 in the formation of cardioprotective effect of ischemic preconditioning. In experiments on isolated rat hearts we show here an increase in the level of UCP2 and UCP3 gene expression in the heart tissue under the influence of ischemic preconditioning with three episodes of 5 min stopping the flow perfusion. Similar effects were induced by a prolonged ischemia-reperfusion of myocardium. The blockade of the UCP2 activity by genipin (Wako Inc., USA, 10(-5) mol/L, isolated heart perfusion for 15 minutes) abolished the protective effect of adaptation to ischemia. It was concluded that uncoupling proteins take part in the cardioprotective effect ofischemic preconditioning.
Asunto(s)
Canales Iónicos/antagonistas & inhibidores , Iridoides/farmacología , Precondicionamiento Isquémico Miocárdico , Proteínas Mitocondriales/antagonistas & inhibidores , Daño por Reperfusión Miocárdica/metabolismo , Daño por Reperfusión Miocárdica/prevención & control , Adaptación Fisiológica/efectos de los fármacos , Adaptación Fisiológica/fisiología , Animales , Técnicas In Vitro , Canales Iónicos/genética , Canales Iónicos/fisiología , Masculino , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/fisiología , Daño por Reperfusión Miocárdica/fisiopatología , Ratas , Ratas Wistar , Proteína Desacopladora 2 , Proteína Desacopladora 3RESUMEN
The influence of mitochondrial permeability transition pore (MPTP) opening on reactive oxygen species (ROS) production in the rat brain mitochondria was studied. It was shown that ROS production is regulated differently by the rate of oxygen consumption and membrane potential, dependent on steady-state or non-equilibrium conditions. Under steady-state conditions, at constant rate of Ca2+-cycling and oxygen consumption, ROS production is potential-dependent and decreases with the inhibition of respiration and mitochondrial depolarization. The constant rate of ROS release is in accord with proportional dependence of the rate of ROS formation on that of oxygen consumption. On the contrary, transition to non-equilibrium state, due to the release of cytochrome c from mitochondria and progressive respiration inhibition, results in the loss of proportionality in the rate of ROS production on the rate of respiration and an exponential rise of ROS production with time, independent of membrane potential. Independent of steady-state or non-equilibrium conditions, the rate of ROS formation is controlled by the rate of potential-dependent uptake of Ca2+ which is the rate-limiting step in ROS production. It was shown that MPTP opening differently regulates ROS production, dependent on Ca2+ concentration. At low calcium MPTP opening results in the decrease in ROS production because of partial mitochondrial depolarization, in spite of sustained increase in oxygen consumption rate by a cyclosporine A-sensitive component due to simultaneous work of Ca2+-uniporter and MPTP as Ca2+-influx and efflux pathways. The effect of MPTP opening at low Ca2+ concentrations is similar to that of Ca2+-ionophore, A-23187. At high calcium MPTP opening results in the increase of ROS release due to the rapid transition to non-equilibrium state because of cytochrome c loss and progressive gating of electron flow in respiratory chain. Thus, under physiological conditions MPTP opening at low intracellular calcium could attenuate oxidative damage and the impairment of neuronal functions by diminishing ROS formation in mitochondria.
Asunto(s)
Encéfalo/metabolismo , Mitocondrias/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Membranas Mitocondriales/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Animales , Calcio/metabolismo , Permeabilidad de la Membrana Celular , Potencial de la Membrana Mitocondrial , Proteínas de Transporte de Membrana Mitocondrial/antagonistas & inhibidores , Poro de Transición de la Permeabilidad Mitocondrial , Consumo de Oxígeno , RatasRESUMEN
We investigated mRNA and protein expression of voltage-dependent anion channel (VDAC), mRNA adenine nucleotide translocase (ANT) as well as the sensitivity of the mitochondrial permeability transition pore opening (MPTP) to Ca2+ in the adult and old rat heart. It was shown that in the old rats hearts VDAC mRNA expression increased by 1.7 (p < 0.05) times and mRNA ANT expression increased by 1.8 (p < 0.05) times in comparison with adult animals. The Western Blot analysis showed that the level of VDAC protein expression in the old rat hearts also significantly increased compared with adult animals. In the hearts of old rats, the sensitivity of MPTP opening to calcium (10(-7)-10(-4) mol/l) determined by mitochondria swelling, increased two-fold (p < 0.05). Therefore, an increased VDAC and ANT expression, as the main structural functional components of the MPTP, and an increased sensitivity of MPTP opening to Ca2+ caused an increase in the permeability of mitochondrial membranes in aging. Each of these factors may contribute to alterations in mitochondrial barrier properties and lead to mitochondrial dysfunction.