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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.

We studied the sensitivity of mitochondrial permeability transition pore (MPTP) opening to natural inductors--Ca2+ ions in the rat heart mitochondria with chronic deficiency of nigrostriatal dopamine caused by an injection of selective neurotoxin 6-hydroxidofamine in an ascending lateral bundle of the forebrain. MPTP-opening was determined specrophotometrically (lamda=520 nm) by a decrease in an optical density resulting from mitochondrial swelling. It has been shown that the rat heart mitochondria with chronic deficiency of nigrostriatal dopamine are more sensitive to Ca2+ in its physiological concentration (10(-7) mol/l) and overload (10(-6) - 10(-4) mol/l) in comparison to control animals. Thus, obtained results lead to a conclusion that an increased sensitivity of the mitochondrial permeability transition pore to calcium and mitochondrial membrane permeability may be one of the causes previously reported of disturbance in contractile function of the rat heart with chronic deficiency of nigrostriatal dopamine.

Nitric oxide reacts rapidly with superoxide to produce the potent oxidant peroxynitrite. In vivo mitochondria produce superoxide as well as NO. In heart mitochondria of aging rats the amount of NO and O2(-) are increased thus the levels of peroxynitrite produced may be increased too, in this reason mitochondria may be a major site of peroxynitrite formation. Oxidative stress induces cyclosporine A-sensitive mitochondrial efflux of calcium and proapoptotic factors through MPTP (mitochondrial permeability transition pore) opening in heart mitochondria which may contribute to tissue damage and mitochondrial dysfunction in aging rats. We tested the levels of NO and superoxide generation in mitochondria simultaneously with cyclosporine A-sensitive MPTP opening by Ca2+ and phenylarsine oxide (PAO) to determine whether downregulation of both NO and O2(-) generation in heart mitochondria by potent steroid antioxidant and free radical scavenger ecdysterone may protect heart mitochondria of aging rats again tissue damage. C27-phytosteroid hormone ecdysterone (10 mkg/100g, per os, 2 weeks) mimics action of its structural analog C27- steroid hormone calcitriol (1alpha,25-dihydroxyvitamin D3) and exert its cardio protection in aging heart mitochondria by inhibition of MPTP opening with effectivity of action of hormone melatonine (150 mkg/100g, 2 weeks [ V.F. Sagach et al. Fyziol. J (Ukr), 2006, 52(2), 3-15]). MPTP inhibition is dependent on paradoxycally high activation by ecdusterone of oxidative degradation of L-arginine by mtcNOS in mitochondria, by downregulation of superoxide generation and L-arginine degradation by arginase II and NO generation by mtiNOS in de novo and by NADP-dependent mtNR (nitrate reductase) in salvage pathways. These results suggest that MPTP opening may be directly influenced by ecdysterone signaling in mitochondria. The signaling pathway by which ecdysterone may coregulate the O2(-) and NO generation in heart mitochondria of aging rats may involve an outer mitochondrial membrane estrogen receptor coupled to mitochondrial PI3K/Akt/PKB activation results in superactivation and constitutive NO synthesis by mtcNOS.

Protective properties of coenzyme Q10 (CoQ10) on the: (i) Langendorff isolated guinea pig heart's function under ischemia and reperfusion (I/R) and on the isolated mitochondria (ii) the mitochondrial permeability transition pore (MPTP) opening under exposure to calcium as natural MPTP inductor and phenylarsine oxide as oxidant--were studied. Physiological characteristic of contractile function, myocardial oxygen consumption and mitochondrial factor release as index of MPTP opening were compared before and after ischemia of isolated heart in control animals and animals with preliminary administration of CoQ10 per os. It have been shown that I/R disturbances of heart function were decreased and oxygen metabolism was normalised in animals treated with CoQ10 in compare to non-treated control. It was accompanied with substantial stabilization of mitochondrial membrane. Decreased I/R disturbances of isolated heart from CoQ10-treated animals were correlated to amount of mitochondrial factor released to coronary flow. Moreover, preliminary incubation of mitochondria, isolated from rat heart, with CoQ10 (10(-5) mol/l) substantially prevented calcium and phenylarsine-induced, cyclosporine A-sensitive mitochondrial swelling. This protective effect was increased in experiments with deenergizing mitochondria. Results of physiological and biochemical study reveal that one of the mechanisms of CoQ10's cardioprotective effect could be direct inhibition of mitochondrial permeability transition pore opening during ischemia and reperfusion of the heart.

This study evaluated generation of O2*- and NO in heart mitochondria isolated from 9-week old streptozotocin (STZ)-induced diabetic rats and the effect of ecdysterone treatment on these parameters. Mitochondria isolated from 9-week old placebo-treated rats were used as control. Several parameters were evaluated: O2*- production, the levels of stable NO metabolites nitrate, nitrite and total nitrosothiols, the level of bilirubine (as marker of CO generation), inducible (iNOS) and constitutive (nNOS) mtNOS, NADH- dependent nitrate reductase (NR) and inducible arginase II (AII) activity. We observed that diabetes was accompanied by a significant decrease in nNOS activity, nitrite, total nitrosothiols and bilirubine content while iNOS, NR and AII activity, as well as O2*- generation was increased in heart mitochondria. Ecdysterone treatment normalized the levels of stable NO metabolites, ability to generate superoxide, iNOS and nNOS activity, but not bilirubine level, NR and AII activity. These results suggest that ecdysterone treatment attenuates diabetes-induced mitochondrial alterations protecting against oxidative and nitrosative stresses. Thus, ecdysterone therapy, besides its well known importance in the maintenance of glycemic control, may help to protect against mitochondrial dysfunction associated to several age-related disorders.

The effect of the hormone of pineal gland melatonin on the ischemic tolerance and the sensitivity of mitochondrial permeability transition pore opening in old rat heart were studied. It has been shown in the Langendorf's isolated rat heart that heart contractile functional changes under ischemia and reperfusion were more pronounced in old (24-27 months) rat hearts in comparison with the adult (5-6 months) animals. A two-week's in vivo course of intraperitoneal injection of melatonin (1,5 mg/kg weight) to old rats contributed to the rehabilitation of the functional changes of isolated heart after ischemia during reperfusion and decreased the sensitivity of mitochondrial permeability transition pore opening to Ca2+ and phenilarsinoxide in comparison with old animals which did not received melatonin. It was accompanied by the significant decreasing in mRNA bax expression in old rat heart, lessening in content of the superoxide radicals and dien conjugates and twice increasing in the activity of constitutive NO-synthase in heart mitochondria of old rat after a course of melatonin injection. The protective feffect of melatonin on the mitochondrial permeability transition pore opening could be used for correction of the cardiac dysfunction with aging.

On the mitochondria isolated from the heart tissue of adult rats we studied the sensitivity of mitochondrial permeability transition pore (MPTP) opening to its inductor--phenylarsine oxide (PAO) after mitochondrial swelling, registered by spectrophotometric technique at n = 520 nm. In adult rat under influence of two modes of normobaric intermittent hypoxic training (IHT): i) softer but prolonged one induced by breathing in normobaric chamber with 11% O2 gas mixture, 15 minuets sessions with 15 minuets rest intervals, 5 times daily (first mode) and ii) more severe but shorter one induced by breathing with 8% O2 gas mixture (second mode) were used. The intensity of lipid peroxidation and antioxidant defense mechanisms in rat organism were estimated before and after IHT by measuring malon dialdehyde (MDA) content and enzymatic activity of superoxide dismutase (SOD) and catalase (CAT) in the blood and the liver. It has been shown that IHT in the first mode didn't essentially influence both on PAO induced, cyclosporin A--sensitive mitochondrial swelling and indexes of lipid peroxidation as well as the SOD and CAT enzymatic activity. It was established that IHT in the second mode caused pronounced increase in MDA content both in the blood and the liver by 67% and 32% respectively; considerable augmentation of SOD activity in this tissues (by 49% and 32% respectively) and CAT activity (by 18% and 43% respectively). Moreover, in forty five days the activity of SOD exceeded its initial level in three times in both the blood and the liver. It has been established that IHT in the second mode provoke to twice decrease in PAO-induced mitochondrial swelling as compared with mitochondria of the control group, and even in forty five days after IHT stopping the protective effect on mitochondrial PTP opening was well-preserved. These effects were completely abolished in the presence of an inhibitor--cyclosporin A (10(-5) mol/l) that demonstrated mitochondrial swelling to be due to the mitochondrial PTP opening. Our experiments showed that the influence of IHT in more severe mode decreased the sensitivity of mitochondria to the PAO in rat heart mitochondria. Thus resistance of the mitochondrial membrane to an inductor of PTP opening--PAO increase under the influence of IHT in the second mode.

An age-related increase in the sensitivity of the mitochondrial permeability transition pore (MPTP) to inductors of it's opening, Ca2+ ions and phenylarsineoxide (PAO) was studied in experiments in vitro on isolated heart mitochondria of adult and old rats. Two indices were measured spectrophotometrically (lambda = 520 nm) by a decrease in an optical density (OD), resulting from mitochondrial swelling and a release of mitochondrial unidentified substances (mitochondrial factor, MF) registered also spectrophotometrically in a range of waves lambda = 230-260 nm. Dose-dependent effect of Ca2+ (10(-7)-10(-4) mol/l) and PAO (10(-8)-10(-4) mol/l) on swelling of the mitochondria were observed in samples from both adult and old rats. Swelling of the mitochondria from the heart of old rats induced by application of the above inductors was more intensive than the respective effect in samples from adult rats. In samples from the heart of both adult and old rats Ca2+ ions within the tested concentration range (10(-7)-10(-4) mol/l) evoked the release of MF in a dose-dependent manner. Mitochondria from the heart of old rats were found to be capable of releasing some amounts of MF in the absence of the MPTP inductors PAO. When this inductor was applied in a 10(-9) to 10(-4) mol/l concentration range, isolated mitochondria from the heart of old rats released unidentified substances with the absorption peaks at two wavelength, lambda = 230 nm and lambda = 240-245 nm. The former peak was found to be Cyclosporin A-insensitive, while the latter peak could be practically completely inhibited by this antibiotic. The concentrations of tested solutions (10(-7) mol/l CaCl2 and 10(-9) mol/l PAO), at which the release of the factor from the mitochondria of the old rat heart was observed, were significantly lower than those in adult rats. Our experimental data show that mitochondria isolated from the heart tissue of old rats demonstrate significantly higher sensitivity to inductors of MPTP-opening, Ca(2+)-overload and PAO as compared to that typical of adult animals. A higher sensitivity of MPTP-opening in the heart of old rats was accompanied by a higher basal level of expression of mRNA of the bax gene, as compared to that found in adult animals. The expression of the bcl-2 gene showed no age group-related differences. It can be supposed that a proapoptotic agent, the Bax protein, is related to an increase in the sensitivity of the MPTP (in particular to that manifested in the processes of pore formation) in the course of aging. Antioxidants, melathonin and trolox, when applied in 10(-5) mol/l concentration, presented to a certain extent opening of the MPTP-induced by 10(-5) mol/l PAO in samples from adult and old rats. These findings can be used for correction of increased sensitivity of the MPTP to different inductors, which is typical of old rats. We conclude that physiological aging is accompanied by the mitochondrial dysfunction. The MF-released capability of the mitochondria from heart tissue of old rats observed both in the presence and absence of MPTP-opening inductors (probably related to a higher sensitivity of MPTP-opening) is one of the manifestation of such dysfunction.

We studied the sensitivity of mitochondrial permeability transition pore (MPTP) opening to its inductor-phenylarsineoxide (PAO) in adult (6 months), old rat heart (24 months) and in old rat heart under conditions of intermittent hypoxic training (IHT). We defined the sensitivity of MPTP opening on two parameters: the alterations in mitochondrial swelling and the release ofmitochondrial substances (mitochondrial factor). It was shown that mitochondria of old rat heart are more sensitive to PAO which caused opening of cyclosporin-sensitive MPTP and MPTP-dependent factor release, in comparison with those of adult rat heart mitochondria. One of the causes of increased sensitivity of MPTP opening to PAO is development of an oxidative stress with age that was accompanied by increase of an active metabolite of oxygen IHT on PAO-induced MPTP-opening and MPTP-dependent factor release from old rat heart mitochondria. IHT also reduced the content of H2O2 and *OH in old rat hearts. IHT can be used as protective procedure preventing MPTP opening in aging and, probably, in a numerous chronic pathological states of organism under oxidative stress.

In experiments in vitro on isolated heart mitochondria of rats, an activation of mitochondrial permeability transition pore (PTP) was induced by either modelling an oxidative stress with PTP-inductor phenylarsine oxide (PAO) or by calcium overload (CaCl2). PTP-opening was determined spectrophotometrically (lambda = 520 nm) by a decrease in an optical density (OD), resulting from mitochondrial swelling. We also observed a release of mitochondrial unidentified substances (mitochondrial factor, MF) registered spectrophotometrically in a range of waves lambda = 230-260 nm. Both correlation between mitochondrial swelling and a release of the mitochondrial factor have been found in experiments with PAO at concentrations 10(-7)-10(-4) mol/l, and in those with CaCl2 at concentrations 10(-6)-10(-4) mol/l. The classical inhibitor of mitochondrial PTP cyclosporin A (Cs A, 10(-5) mol/l) inhibited mitochondrial swelling and a release of that factor completely. Our experimental data give evidence for mitochondrial origin of the factor and its release following PTP-opening by PTP-inductors--PAO and CaCl2. Mitochondrial swelling that accompanied the factor's release might contribute to PTP-opening and be useful in defining the mitochondrial sensitivity either with inductors or inhibitors of mitochondrial PTP in different tissues under normal and pathological states of organism.