Redox State Of Pyridine Nucleotides Research Articles

Integrated redox sensitivity of vascular Kv1 channels

Voltage‐gated potassium (Kv) channels in vascular smooth muscle are responsive to a range of molecular signals that link local metabolic demand to blood supply. We recently found that elevation of local lactate:pyruvate, reflective of enhanced tissue oxygen demand and consequent changes in smooth muscle cytosolic NADH:NAD+, potentiate native Kv1 activity in a manner that is dependent on pore complex‐associated Kvβ proteins (i.e., Kvβ1 and Kvβ2). Considering that these proteins also participate in the regulation of Kv function in response to oxidants, we tested the hypothesis that Kv1‐dependent vasoreactivity to the endogenous dilator hydrogen peroxide (H2O2) is modified by intracellular NAD(H) redox status. Application of H2O2 to preconstricted (100 nM U46619, 80 mmHg intravascular pressure) mesenteric arteries from mice resulted in concentration‐dependent vasodilation (0.1‐10 μΜ). However, the magnitude of vasodilation in response to 10 μM H2O2 was significantly enhanced when applied in the presence of 10 mM L‐lactate (10 μΜ Η2Ο2: 16 ± 9%; 10 mM L‐lactate + 10 μM H2O2: 80 ± 15% dilation), but not in the presence of 10 mM pyruvate (~11% H2O2‐induced constriction). Consistent with direct effects of pyridine nucleotide redox state on oxidant‐induced Kv potentiation, increases in single Kv1 channel open probability in excised membrane patches from isolated vascular smooth muscle cells in response to 10 μM H2O2 were significantly enhanced in the presence of 1 mM NADH. Based on these results, we examined the role of the intracellular Kvβ subunit complex in redox‐modified vasodilation in response to H2O2. Arteries isolated from double transgenic mice with doxycycline‐inducible increases in smooth muscle Kvβ1:Kvβ2 protein ratio exhibited a complete loss of L‐lactate‐induced enhancement of vasodilation to H2O2. Together, our results suggest that the Kvβ subunit complex in vascular smooth muscle may integrate multiple redox signals to enable tight control over Kv1 gating, vascular tone, and blood flow upon dynamic changes in metabolic demand.

Pyridine nucleotide redox potential in coronary smooth muscle couples myocardial blood flow to cardiac metabolism

Adequate oxygen delivery to the heart during stress is essential for sustaining cardiac function. Acute increases in myocardial oxygen demand evoke coronary vasodilation and enhance perfusion via functional upregulation of smooth muscle voltage-gated K+ (Kv) channels. Because this response is controlled by Kv1 accessory subunits (i.e., Kvβ), which are NAD(P)(H)-dependent aldo-keto reductases, we tested the hypothesis that oxygen demand modifies arterial [NAD(H)]i, and that resultant cytosolic pyridine nucleotide redox state influences Kv1 activity. High-resolution imaging mass spectrometry and live-cell imaging reveal cardiac workload-dependent increases in NADH:NAD+ in intramyocardial arterial myocytes. Intracellular NAD(P)(H) redox ratios reflecting elevated oxygen demand potentiate native coronary Kv1 activity in a Kvβ2-dependent manner. Ablation of Kvβ2 catalysis suppresses redox-dependent increases in Kv1 activity, vasodilation, and the relationship between cardiac workload and myocardial blood flow. Collectively, this work suggests that the pyridine nucleotide sensitivity and enzymatic activity of Kvβ2 controls coronary vasoreactivity and myocardial blood flow during metabolic stress.

The level of secondary messengers and the redox state of NAD+/NADH are associated with sperm quality in infertility

In order to explore the interrelation of Calcium, cAMP, and redox state of pyridine nucleotides in seminal plasma and ejaculate quality in cases of idiopathic infertility we conducted an evaluation of 170 infertile males and 46 fertile males aged 20–43 years. Sperm analysis was undertaken according to WHO protocol. The content of Calcium in the seminal plasma was detected using optical emission spectrometry, cAMP levels were determined via enzymatic immunoassay. The redox state of pyridine nucleotides was evaluated from the ratio of pyruvate to lactate, determined via enzymatic method. Our results show a decrease in Calcium, cAMP, pyruvate and the oxidation-reduction potential of pyridine nucleotides in the seminal plasma of infertile males with pathospermia. This corresponds to anaerobic inversion of oxidative conversions and metabolism inadaptation. Such processes are often seen in inflammatory and autoimmune conditions. cAMP levels reliably correlated with the number of progressively mobile sperm cells, but not with the number of their pathological forms. A positive correlation between the concentration of cAMP and calcium was discovered as well. Pathospermia was characterized by the positive relation between the value of the NAD+/NADH coefficient and the spermatozoa concentration that was not present in fertile donors. Our study shows distinct changes in the concentration of secondary messengers and redox state of pyridine nucleotides in the seminal fluid that can act as molecular predictors for the development of idiopathic infertility.

Effect of detonation nanodiamond surface composition on physiological indicators of mitochondrial functions

For the first time, an effect of detonation nanodiamonds (NDs) with different surface compositions on the main functional characteristics of isolated rat liver mitochondria was studied. The response of membrane potential, calcium retention capacity, and redox state of pyridine nucleotides have been monitored upon the administration of NDs functionalized with carboxyl, hydroxyl, amine, hydrogen, and chlorine surface groups. Hydrogenated and chlorinated NDs caused reduction of the membrane potential and calcium retention capacity of mitochondria. An aminated ND caused an even greater decrease in calcium retention capacity (at a concentration of 0.75 mg/ml), reducing it to 65% of the control. The use of cyclosporine A prevented a decrease in membrane potential and calcium retention capacity indicating the induction of non-specific mitochondrial membrane pores during the NDs incubation with mitochondria. Hydrogenated and chlorinated NDs had no significant effect on the redox state of mitochondrial pyridine nucleotides. Other NDs studied had no effects on functional characteristics of mitochondria, even at high concentrations (up to 1.5 mg/ml). High activity of chlorinated and hydrogenated NDs may be due to the greater hydrophobicity of their surface and its interaction with mitochondrial pores components. Thus, isolated rat liver mitochondria can be used as a biomodel for initial testing of ND samples to assess the possibility of their use in drug delivery systems.

Cortisol promotes endoplasmic glucose production via pyridine nucleotide redox.

Both increased adrenal and peripheral cortisol production, the latter governed by 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1), contribute to the maintenance of fasting blood glucose. In the endoplasmic reticulum (ER), the pyridine nucleotide redox state (NADP/NADPH) is dictated by the concentration of glucose-6-phosphate (G6P) and the coordinated activities of two enzymes, hexose-6-phosphate dehydrogenase (H6PDH) and 11β-HSD1. However, luminal G6P may similarly serve as a substrate for hepatic glucose-6-phophatase (G6Pase). A tacit belief is that the G6P pool in the ER is equally accessible to both H6PDH and G6Pase. Based on our inhibition studies and kinetic analysis in isolated rat liver microsomes, these two aforesaid luminal enzymes do share the G6P pool in the ER, but not equally. Based on the kinetic modeling of G6P flux, the ER transporter for G6P (T1) preferentially delivers this substrate to G6Pase; hence, the luminal enzymes do not share G6P equally. Moreover, cortisol, acting through 11β-HSD1, begets a more reduced pyridine redox ratio. By altering this luminal redox ratio, G6P flux through H6PDH is restrained, allowing more G6P for the competing enzyme G6Pase. And, at low G6P concentrations in the ER lumen, which occur during fasting, this acute cortisol-induced redox adjustment promotes glucose production. This reproducible cortisol-driven mechanism has been heretofore unrecognized.

Aluminium effects on pyridine nucleotide redox state in roots of Scots pine

After prolonged (3-9 weeks) hydroponic treatment of Scots pine seedlings with different concentrations (0.5-4.0 mM) of Al (AI(N0<sub>3</sub>)<sub>3</sub>), the levels of pyridine nucleotides were determined in root homogenates. After 3 weeks of Al stress, a significant decrease of the anabolic reduction charge (ARC: NADPH/(NADP<sup>+</sup> + NADPH)) and an increase of the redox status (NAD(P)H/NAD(P)<sup>+</sup>), catabolic reduction charge (CRC: NADH/(NAD<sup>+</sup> + NADH)) and phosphorylation capacity expressed as NADP<sup>+</sup>/NAD<sup>+</sup> ratio was found in the 4.0 mM Al treatment. After 6 weeks, Al at concentrations of 0.5 and 1.0 mM induced an enhancement of the NADH level and a reduction of NADPH level, but the redox ratios were not changed significantly. After 9 weeks treatment with Al concentrations ranging from 0.5 to 4.0 mM, decreases of the relative level of NADP<sup>+</sup>, NADPH and NADH and increases of NAD<sup>+</sup> were found. Consequently, the CRC, NAD(P)H/NAD(P)<sup>+</sup> and NADP<sup>+</sup>/NAD<sup>+</sup> ratios reached a minimum and ARC a maximum as compared to previous measurements.

Regulation of the Na+/Ca2+ Exchanger by Pyridine Nucleotide Redox Potential in Ventricular Myocytes

The cardiac Na(+)/Ca(2+) exchanger (NCX) is the major Ca(2+) efflux pathway on the sarcolemma, counterbalancing Ca(2+) influx via L-type Ca(2+) current during excitation-contraction coupling. Altered NCX activity modulates the sarcoplastic reticulum Ca(2+) load and can contribute to abnormal Ca(2+) handling and arrhythmias. NADH/NAD(+) is the main redox couple controlling mitochondrial energy production, glycolysis, and other redox reactions. Here, we tested whether cytosolic NADH/NAD(+) redox potential regulates NCX activity in adult cardiomyocytes. NCX current (INCX), measured with whole cell patch clamp, was inhibited in response to cytosolic NADH loaded directly via pipette or increased by extracellular lactate perfusion, whereas an increase of mitochondrial NADH had no effect. Reactive oxygen species (ROS) accumulation was enhanced by increasing cytosolic NADH, and NADH-induced INCX inhibition was abolished by the H2O2 scavenger catalase. NADH-induced ROS accumulation was independent of mitochondrial respiration (rotenone-insensitive) but was inhibited by the flavoenzyme blocker diphenylene iodonium. NADPH oxidase was ruled out as the effector because INCX was insensitive to cytosolic NADPH, and NADH-induced ROS and INCX inhibition were not abrogated by the specific NADPH oxidase inhibitor gp91ds-tat. This study reveals a novel mechanism of NCX regulation by cytosolic NADH/NAD(+) redox potential through a ROS-generating NADH-driven flavoprotein oxidase. The mechanism is likely to play a key role in Ca(2+) homeostasis and the response to alterations in the cytosolic pyridine nucleotide redox state during ischemia-reperfusion or other cardiovascular diseases.

Regulation of Na+/CA2+ Exchanger by Pyridine Nucleotide Redox Potential in Cardiac Myocytes

Cardiac Na+/Ca2+ exchanger (NCX) is the major Ca2+ efflux pathway on the sarcolemmal membrane, counterbalancing Ca2+ influx via L-type Ca2+ current during excitation-contraction coupling. In humans, guinea pigs and large mammals, NCX removes 20-25% of total cytosolic Ca2+ during relaxation. Therefore, altered NCX activity can modulate SR Ca2+ load and contribute to abnormal Ca2+ handling and arrhythmias. Here, we demonstrate that: 1) increased NADH inhibits NCX current (INCX); 2) NCX inhibition by NADH is reactive oxygen species (ROS)-dependent; 3) the NADH-induced ROS is independent of the mitochondrial electron transportation chain (ETC). INCX was measured with whole cell patch-clamp at 37°C after dialyzing the cell with various pipette solutions. With 400µM NADH in the pipette solution, INCX (at +80mV) was reduced by 65% compared to control. Endogenously elevated cytosolic NADH by lactate perfusion (1mM) also inhibited INCX by 79% (at +80mV) compared to control without lactate perfusion. Measurement of ROS revealed that increasing cytosolic NADH increased ROS accumulation. The NADH-induced increase in ROS, as well as the inhibition of INCX, was abrogated by co-dialysis with the H2O2 scavenger catalase, demonstrating that NCX inhibition is mediated by ROS. Inhibition of ETC with rotenone did not reverse NCX inhibition by NADH, whereas D diphenylene iodonium (DPI), which inhibits both the ETC and NAD(P)H oxidases, abolished NCX inhibition. Lactate also induced ROS production in cell suspensions in a dose-dependent manner, which was inhibited by DPI but not by rotenone, NaCN, or antimycin A. Our findings indicate that NCX is regulated by cytosolic NADH/NAD+ redox status through a ROS-dependent mechanism involving NAD(P)H oxidase, a mechanism that is likely to have profound effects on Ca2+ homeostasis under conditions that alter cytosolic pyridine nucleotide redox state, for example, during ischemia-reperfusion, diabetes or chronic heart failure.

Production of H2O2 in the Endoplasmic Reticulum Promotes In Vivo Disulfide Bond Formation

Oxidative protein folding in the luminal compartment of endoplasmic reticulum (ER) is thought to be accompanied by the generation of H₂O₂, as side-product of disulfide bond formation. We aimed to examine the role of H₂O₂ produced in the lumen, which on one hand can lead to redox imbalance and hence can contribute to ER stress caused by overproduction of secretory proteins; on the other hand, as an excellent electron acceptor, H₂O₂ might serve as an additional pro-oxidant in physiological oxidative folding. Stimulation of H₂O₂ production in the hepatic ER resulted in a decrease in microsomal GSH and protein-thiol contents and in a redox shift of certain luminal oxidoreductases in mice. The oxidative effect, accompanied by moderate signs of ER stress and reversible dilation of ER cisternae, was prevented by concomitant reducing treatment. The imbalance also affected the redox state of pyridine nucleotides in the ER. Antibody producing cells artificially engineered with powerful luminal H₂O₂ eliminating system showed diminished secretion of mature antibody polymers, while incomplete antibody monomers/dimers were accumulated and/or secreted. Evidence are provided by using in vivo models that hydrogen peroxide can promote disulfide bond formation in the ER. The results indicate that local H₂O₂ production promotes, while quenching of H₂O₂ impairs disulfide formation. The contribution of H₂O₂ to disulfide bond formation previously observed in vitro can be also shown in cellular and in vivo systems.

Decreased prereceptorial glucocorticoid activating capacity in starvation due to an oxidative shift of pyridine nucleotides in the endoplasmic reticulum

Redox state of pyridine nucleotides of the endoplasmic reticulum (ER) lumen was determined in different nutritional conditions. NADPH-dependent cortisone reduction and NADP +-dependent cortisol oxidation were measured in rat liver microsomes, by utilizing the luminal 11β-hydroxysteroid dehydrogenase type 1 activity. Cortisone reduction decreased, while cortisol oxidation increased during onward starvation, showing that the luminal NADPH/NADP + ratio was substantially decreased. Cortisone or metyrapone addition caused a smaller decrease in NADPH fluorescence in microsomes from starved rats. The results demonstrate that nutrient supply is mirrored by the redox state of ER luminal pyridine nucleotides.

Reactive γ-ketoaldehydes formed via the isoprostane pathway disrupt mitochondrial respiration and calcium homeostasis

Isoketals (IsoKs) are γ-ketoaldehydes formed via the isoprostane pathway of arachidonic acid peroxidation and are among the most reactive by-products of lipid peroxidation. IsoKs selectively adduct to protein lysine residues and are highly cytotoxic, but the targets and molecular events involved in IsoK-induced cell death are poorly defined. Our previous work established that physiologically relevant aldehydes induce mitochondrial dysfunction (Kristal et al., J. Biol. Chem. 271:6033–6038; 1996). We therefore examined whether IsoKs induced mitochondrial dysfunction. Incubation of mitochondria with synthetic IsoKs in the presence or absence of Ca 2+ was associated with alterations in mitochondrial respiration, membrane potential (ΔΨ), and pyridine nucleotide redox state. IsoKs dose dependently (0.5–4 µM) accelerated liver mitochondria swelling induced by low concentrations of Ca 2+ and Zn 2+ or by the prooxidant tert-butylhydroperoxide , and release of cytochrome c, with similar observations in heart/brain mitochondria. The mitochondrial permeability transition (mPT) inhibitor cyclosporine A delayed IsoK-induced mitochondria dysfunction. The actions of IsoKs are consistent with interactions with cytochrome c, a protein rich in lysine residues. Direct reaction of IsoKs with select lysines in cytochrome c was demonstrated using high-resolution mass spectrometry. Overall, these results suggest that IsoKs may, in part, mediate their cytotoxic effects through induction of the mPT and subsequent activation of downstream cell death cascades.

Altered redox state of luminal pyridine nucleotides facilitates the sensitivity towards oxidative injury and leads to endoplasmic reticulum stress dependent autophagy in HepG2 cells

Maintenance of the reduced state of luminal pyridine nucleotides in the endoplasmic reticulum – an important pro-survival factor in the cell – is ensured by the concerted action of glucose-6-phosphate transporter and hexose-6-phosphate dehydrogenase. The mechanism by which the redox imbalance leads to cell death was investigated in HepG2 cells. The chemical inhibition of the glucose-6-phosphate transporter, the silencing of hexose-6-phosphate dehydrogenase and/or the glucose-6-phosphate transporter, or the oxidation of luminal NADPH by themselves did not cause a significant loss of cell viability. However, these treatments caused ER calcium store depletion. If these treatments were supplemented with the administration of a subliminal dose of the oxidizing agent menadione, endoplasmic reticulum vacuolization and a loss of viability were observed. Combined treatments resulted in the activation of ATF6 and procaspase-4, and in the induction of Grp78 and CHOP. In spite of the presence of UPR markers and proapoptotic signaling the effector caspases – caspase-3 and caspase-7 – were not active. On the other hand, an elevation of the autophagy marker LC3B was observed. Immunohistochemistry revealed a punctuated distribution of LC3B II, coinciding with the vacuolization of the endoplasmic reticulum. The results suggest that altered redox state of endoplasmic reticulum luminal pyridine nucleotides sensitizes the cell to autophagy.

Phosphorylation of CtBP1 by PKA Induces CYP17 by Promoting CtBP Heterooligomerization

In the human adrenal cortex, the peptide hormone adrenocorticotropin (ACTH) directs cortisol and adrenal androgen biosynthesis by activating a cAMP/protein kinase A (PKA) pathway. Carboxy terminal binding protein 1 (CtBP1) is a corepressor that regulates transcription of the CYP17 gene by periodically interacting with steroidogenic factor‐1 (SF‐1) in response to ACTH signaling. Given that CtBP1 function is regulated by NADH binding, we hypothesized that ACTH‐stimulated changes in cellular pyridine nucleotide concentrations modulate the ability of CtBP1 to repress CYP17 transcription. Further, we postulated that PKA evokes changes in the phosphorylation status of CtBP1 that control the ability of the protein to bind to SF‐1 and repress CYP17 gene expression. We show that ACTH alters pyridine nucleotide redox state and identify amino acid residues in CtBP1 that are targeted by PKA and PAK6. Both ACTH/cAMP signaling and NADH/NAD+ ratio stimulate nuclear‐cytoplasmic oscillation of both CtBP proteins. We provide evidence that PKA 1) induces metabolic changes in the adrenal cortex and 2) phosphorylates CtBP proteins, particularly CtBP1 at T144, resulting in ACTH‐dependent CYP17 transcription.

Phosphorylation of CtBP1 by cAMP-dependent Protein Kinase Modulates Induction of CYP17 by Stimulating Partnering of CtBP1 and 2

In the human adrenal cortex, the peptide hormone adrenocorticotropin (ACTH) directs cortisol and adrenal androgen biosynthesis by activating a cAMP/cAMP-dependent protein kinase (PKA) pathway. Carboxyl-terminal binding protein 1 (CtBP1) is a corepressor that regulates transcription of the CYP17 gene by periodically interacting with steroidogenic factor-1 in response to ACTH signaling. Given that CtBP1 function is regulated by NADH binding, we hypothesized that ACTH-stimulated changes in cellular pyridine nucleotide concentrations modulate the ability of CtBP1 to repress CYP17 transcription. Further, we postulated that PKA evokes changes in the phosphorylation status of CtBP1 that control the ability of the protein to bind to steroidogenic factor-1 and the coactivator GCN5 (general control nonderepressed 5) and repress CYP17 gene expression. We show that ACTH alters pyridine nucleotide redox state and identify amino acid residues in CtBP1 that are targeted by PKA and PAK6. Both ACTH/cAMP signaling and NADH/NAD+ ratio stimulate nuclear-cytoplasmic oscillation of both CtBP proteins. We provide evidence that PKA 1) induces metabolic changes in the adrenal cortex and 2) phosphorylates CtBP proteins, particularly CtBP1 at T144, resulting in CtBP protein partnering and ACTH-dependent CYP17 transcription.

Anoxia-Induced Changes in Pyridine Nucleotide Redox State in Cortical Neurons and Astrocytes

NAD(P)H autofluorescence was used to verify establishment of metabolic anoxia using primary cultures of cortical neurons and astrocytes. Cells on cover slips were placed in a chamber and O(2) was displaced by continuous infusion of argon. Perfusion with medium at PO(2) < 0.4 mm Hg caused an increase in NAD(P)H fluorescence, albeit to levels lower than that obtained with cyanide. Addition of the nitric oxide-generating agent DETA-NO to the hypoxic medium further increased fluorescence to the level with cyanide. Fluorescence under anoxia remained high in the presence of glucose, but declined in neurons and not in astrocytes when glucose was substituted with 2-deoxyglucose. Reoxygenation of neurons resulted in a decline in fluorescence and a loss in fluorescent gradient between fully reduced and fully oxidized (plus respiratory uncoupler). We conclude that (1) DETA-NO is useful for generating metabolic anoxia in the presence of argon (2) Exogenous glucose is necessary to maintain NAD(P)H in a reduced state during metabolic anoxia in neurons but not astrocytes (3) Neurons undergo a partially irreversible decline in NAD(P)H fluorescence during metabolic anoxia and reoxygenation that could contribute to prolonged metabolic failure.

Uncoupled Redox Systems in the Lumen of the Endoplasmic Reticulum: PYRIDINE NUCLEOTIDES STAY REDUCED IN AN OXIDATIVE ENVIRONMENT

The redox state of the intraluminal pyridine nucleotide pool was investigated in rat liver microsomal vesicles. The vesicles showed cortisone reductase activity in the absence of added reductants, which was dependent on the integrity of the membrane. The intraluminal pyridine nucleotide pool could be oxidized by the addition of cortisone or metyrapone but not of glutathione. On the other hand, intraluminal pyridine nucleotides were slightly reduced by cortisol or glucose 6-phosphate, although glutathione was completely ineffective. Redox state of microsomal protein thiols/disulfides was not altered either by manipulations affecting the redox state of pyridine nucleotides or by the addition of NAD(P)+ or NAD(P)H. The uncoupling of the thiol/disulfide and NAD(P)+/NAD(P)H redox couples was not because of their subcompartmentation, because enzymes responsible for the intraluminal oxidoreduction of pyridine nucleotides were distributed equally in smooth and rough microsomal subfractions. Instead, the phenomenon can be explained by the negligible representation of glutathione reductase in the endoplasmic reticulum lumen. The results demonstrated the separate existence of two redox systems in the endoplasmic reticulum lumen, which explains the contemporary functioning of oxidative folding and of powerful reductive reactions.

Effects of Decreasing Mitochondrial Volume on the Regulation of the Permeability Transition Pore

The permeability transition pore (PTP) is a Ca(2+)-sensitive mitochondrial inner membrane channel involved in several models of cell death. Because the matrix concentration of PTP regulatory factors depends on matrix volume, we have investigated the role of the mitochondrial volume in PTP regulation. By incubating rat liver mitochondria in media of different osmolarity, we found that the Ca(2+) threshold required for PTP opening dramatically increased when mitochondrial volume decreased relative to the standard condition. This shrinkage-induced PTP inhibition was not related to the observed changes in protonmotive force, or pyridine nucleotide redox state and persisted when mitochondria were depleted of adenine nucleotides. On the other hand, mitochondrial volume did not affect PTP regulation when mitochondria were depleted of Mg(2+). By studying the effects of Mg(2+), cyclosporin A (CsA) and ubiquinone 0 (Ub(0)) on PTP regulation, we found that mitochondrial shrinkage increased the efficacy of Mg(2+) and Ub(0) at PTP inhibition, whereas it decreased that of CsA. The ability of mitochondrial volume to alter the activity of several PTP regulators represents a hitherto unrecognized characteristic of the pore that might lead to a new approach for its pharmacological modulation.

The effect of cytoplasmic Ca 2+ signal on the redox state of mitochondrial pyridine nucleotides

As first observed in rat adrenal glomerulosa cells, cytoplasmic Ca 2+ signal, induced by K +, angiotensin II or vasopressin, evokes an increase in the level of reduced mitochondrial pyridine nucleotides, NADH and NADPH. Prostaglandin F 2α and extracellular ATP exert similar effects in rat ovarian luteal cells. This coupling of cytoplasmic Ca 2+ concentration and mitochondrial metabolism occurs also when the stimuli are applied at physiological concentration and under conditions when no formation of high-Ca 2+ perimitochondrial microdomains may be presumed. We present evidence that low submicromolar Ca 2+ signals in the cytoplasm can increase mitochondrial Ca 2+ concentration and activate mitochondrial dehydrogenation processes. Several observations support the assumption that intramitochondrial Ca 2+ signals play a significant role in the stimulation of steroid hormone production.

Mitochondrial thioredoxin reductase inhibition by gold(I) compounds and concurrent stimulation of permeability transition and release of cytochrome c

The effects of auranofin, chloro(triethylphosphine)gold(I) (TEPAu), and aurothiomalate on mitochondrial respiration, pyridine nucleotide redox state, membrane permeability properties, and redox enzymes activities were compared. The three gold(I) derivatives, in the submicromolar range, were extremely potent inhibitors of thioredoxin reductase and stimulators of the mitochondrial membrane permeability transition (MPT). Auranofin appeared as the most effective one. In the micromolar range, it inhibited respiratory chain and glutathione peroxidase activity only slightly if not at all. TEPAu and aurothiomalate exhibited effects similar to auranofin, although TEPAu showed a moderate inhibition on respiration. Aurothiomalate inhibited glutathione peroxidase at concentrations where auranofin and TEPAu were without effect. Under nonswelling conditions, the presence of auranofin and aurothiomalate did not alter the redox properties of the mitochondrial pyridine nucleotides indicating that membrane permeability transition occurred independently of the preliminary oxidation of pyridine nucleotides. Under the same experimental conditions, TEPAu showed a moderate stimulation of pyridine nucleotides oxidation. Mitochondrial total thiol groups, in the presence of the gold(I) derivatives, slightly decreased, indicating the occurrence of an oxidative trend. Concomitantly with MPT, gold(I) compounds determined the release of cytochrome c that, however, occurred also in the presence of cyclosporin A and, partially, of EGTA, indicating its independence of MPT. It is concluded that the specific inhibition of thioredoxin reductase by gold(I) compounds may be the determinant of MPT and the release of cytochrome c.

Biphasic Oxidation of Mitochondrial NAD(P)H

The redox state of mitochondrial pyridine nucleotides is known to be important for structural integrity of mitochondria. In this work, we observed a biphasic oxidation of endogenous NAD(P)H in rat liver mitochondria induced by tert-butylhydroperoxide. Nearly 85% of mitochondrial NAD(P)H was rapidly oxidized during the first phase. The second phase of NAD(P)H oxidation was retarded for several minutes, appearing after the inner membrane potential collapse and mitochondria swelling. It was characterized by disturbance of ATP synthesis and dramatic permeabilization of the inner membrane to pyridine nucleotides. The second phase was completely prevented by 0.5 μM cyclosporin A or 0.2 mM EGTA or was significantly delayed by 25 μM butylhydroxytoluene or trifluoperazine. The obtained data suggest that the second phase resulted from oxidation of the remaining NADH via the outer membrane electron transport system of permeabilized mitochondria, leading to further oxidation of the remaining NADPH in a transhydrogenase reaction.