Reducing Cancer Risk Through Primary Prevention Activities Among Children: A Demonstration Project.

CONTEXT: Opportunities to reduce the risk of cancer, including cervical, liver, and skin cancer, start early in life. To encourage adoption of primary prevention activities in childhood to reduce cancer risk later in life, Centers for Disease Control and Prevention conducted a demonstration project with 3 National Comprehensive Cancer Control Program (NCCCP) recipients. PROGRAM: Iowa, Northwest Portland Area Indian Health Board (NPAIHB), and Pennsylvania NCCCP recipients implemented evidence-based primary prevention activities for cervical, liver, and skin cancer among children using health care provider education, patient education, and policy development. IMPLEMENTATION: Iowa implemented an announcement approach to improve provider education on human papillomavirus (HPV) vaccination. Pennsylvania focused on patient education for reducing skin cancer risk and both provider and patient education for liver cancer prevention. NPAIHB created a sun safety intervention for tribal organizations, including a policy guide, media materials, and patient education. RESULTS: In Iowa, health care providers taking the announcement approach reported significantly higher mean scores on a posttest compared with a pretest regarding perceptions about HPV vaccination, self-efficacy, and behavioral intentions related to vaccination. Pennsylvania integrated sun safety education and sunscreen dispenser programs as a health and wellness initiative in 8 state parks and the Pennsylvania Department of Conservation and Natural Resources incorporated the program in its Pennsylvania Outdoor Recreation Plan. Pennsylvania also implemented health care provider education on the primary prevention of liver cancer through hepatitis B and hepatitis C screening and hepatitis B vaccination. The NPAIHB skin cancer policy guide was created and distributed for use to all 43 federally recognized tribes of Oregon, Washington, and Idaho served by NPAIHB. DISCUSSION: The identification, dissemination, and implementation of these efforts can serve as best practices for future childhood primary prevention programs. NCCCP recipients and public health professionals can use health care provider education, patient education, and policy development to reduce future risk for cervical, liver, and skin cancer among children.

Stress-induced phospho-ubiquitin formation causes parkin degradation.

Mutations in the E3 ubiquitin ligase parkin are the most common known cause of autosomal recessive Parkinson's disease (PD), and parkin depletion may play a role in sporadic PD. Here, we sought to elucidate the mechanisms by which stress decreases parkin protein levels using cultured neuronal cells and the PD-relevant stressor, L-DOPA. We find that L-DOPA causes parkin loss through both oxidative stress-independent and oxidative stress-dependent pathways. Characterization of the latter reveals that it requires both the kinase PINK1 and parkin's interaction with phosphorylated ubiquitin (phospho-Ub) and is mediated by proteasomal degradation. Surprisingly, autoubiquitination and mitophagy do not appear to be required for such loss. In response to stress induced by hydrogen peroxide or CCCP, parkin degradation also requires its association with phospho-Ub, indicating that this mechanism is broadly generalizable. As oxidative stress, metabolic dysfunction and phospho-Ub levels are all elevated in PD, we suggest that these changes may contribute to a loss of parkin expression.

Gene disruption of ribosomal protein L5 (RPL5) decreased the sensitivity of CHO-K1 cells to uncoupler carbonylcyanide-3-chlorophenylhydrazone.

Protonophoric uncoupler carbonylcyanide-3-chlorophenylhydrazone (CCCP) decreases the proton motive force (ΔP) of the mitochondrial inner membrane and results in inhibition of oxidative phosphorylation. In this study, a CCCP-resistant clone was isolated from a random gene trap insertional mutant library of Chinese hamster ovary (CHO)-K1 cells which was constructed by infecting a retrovirus vector, ROSAßgeo. Although we expected the isolation of the mutants defective in nuclear genes responsible for mitochondrial functions, the disrupted gene of the isolated mutant that we named R1 cells was identified as one of the alleles for ribosomal protein 5 of large subunit (RPL5). The R1 cells express as much as 80% RPL5 protein compared with the parental CHO-K1 cells, possibly due to enhanced transcription from a remaining wild-type RPL5 allele in R1 cells. Furthermore, the protein amount is not decreased by CCCP in R1 cells, in contrast to its clear reduction by CCCP in parental cells. Since mutations of RPL5 and other ribosomal proteins are responsible for the ribosomopathies and cancer, the present mutant may be a useful cellular model of such human diseases from a viewpoint of energy metabolism as well as a tool for the study of ribosome biogenesis and extra-ribosomal function of the RPL5 protein.

Pleckstrin homology domain of p210 BCR-ABL interacts with cardiolipin to regulate its mitochondrial translocation and subsequent mitophagy.

Chronic myeloid leukemia (CML) is caused by the chimeric protein p210 BCR-ABL encoded by a gene on the Philadelphia chromosome. Although the kinase domain of p210 BCR-ABL is an active driver of CML, the pathological role of its pleckstrin homology (PH) domain remains unclear. Here, we carried out phospholipid vesicle-binding assays to show that cardiolipin (CL), a characteristic mitochondrial phospholipid, is a unique ligand of the PH domain. Arg726, a basic amino acid in the ligand-binding region, was crucial for ligand recognition. A subset of wild-type p210 BCR-ABL that was transiently expressed in HEK293 cells was dramatically translocated from the cytosol to mitochondria in response to carbonyl cyanide m-chlorophenylhydrazone (CCCP) treatment, which induces mitochondrial depolarization and subsequent externalization of CL to the organelle's outer membrane, whereas an R726A mutant of the protein was not translocated. Furthermore, only wild-type p210 BCR-ABL, but not the R726A mutant, suppressed CCCP-induced mitophagy and subsequently enhanced reactive oxygen species production. Thus, p210 BCR-ABL can change its intracellular localization via interactions between the PH domain and CL to cope with mitochondrial damage. This suggests that p210 BCR-ABL could have beneficial effects for cancer proliferation, providing new insight into the PH domain's contribution to CML pathogenesis.

Effect of Cyanide on Mitochondrial Membrane Depolarization Induced by Uncouplers.

In this work, it was found that the ability of common uncouplers - carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) and 2,4-dinitrophenol (DNP) - to reduce membrane potential of isolated rat liver mitochondria was diminished in the presence of millimolar concentrations of the known cytochrome c oxidase inhibitor - cyanide. In the experiments, mitochondria were energized by addition of ATP in the presence of rotenone, inhibiting oxidation of endogenous substrates via respiratory complex I. Cyanide also reduced the uncoupling effect of FCCP and DNP on mitochondria energized by succinate in the presence of ferricyanide. Importantly, cyanide did not alter the protonophoric activity of FCCP and DNP in artificial bilayer lipid membranes. The causes of the effect of cyanide on the efficiency of protonophoric uncouplers in mitochondria are considered in the framework of the suggestion that conformational changes of membrane proteins could affect the state of lipids in their vicinity. In particular, changes in local microviscosity and vacuum permittivity could change the efficiency of protonophore-mediated translocation.

Differential effects of mitochondrial inhibitors on porcine granulosa cells and oocytes.

Oocytes and granulosa cells rely primarily on mitochondrial respiration and glycolysis for energy production, respectively. The present study examined the effect of mitochondrial inhibitors on the ATP contents of oocytes and granulosa cells. Cumulus cell-oocyte complexes (COCs) and granulosa cells (GCs) were collected from the antral follicles of porcine ovaries. Treatment of denuded oocytes with either carbonyl cyanide m-chlorophenyl hydrazine (CCCP), antimycin, or oligomycin significantly reduced ATP content to very low levels (CCCP, 0.12 pM; antimycin, 0.07 pM; and oligomycin, 0.25 pM; P < 0.05), whereas treatment with a glycolysis inhibitor (bromopyruvic acid, BA) had no effect. Conversely, the ATP content of granulosa cells was significantly reduced by treatment with the glycolysis inhibitor but was not affected by the mitochondrial inhibitors (ATP/10,000 cells; control, 1.78 pM and BA, 0.32 pM; P < 0.05). Reactive oxygen species (ROS) generation after CCCP treatment was greater in oocytes (1.6-fold) than that seen in granulosa cells (1.08-fold). Oocytes surrounded by granulosa cells had higher ATP levels than denuded oocytes. Treatment of COCs with CCCP reduced, but did not completely abolish, ATP content in oocytes (control, 3.15 pM and CCCP, 0.52 pM; P < 0.05), whereas treatment with CCCP plus a gap junction inhibitor, 18α-glycyrrhetinic acid, and CCCP decreased the ATP content to even lower levels (0.29 pM; P < 0.05). These results suggest that granulosa cells are dependent on glycolysis and provide energy to oocytes through gap junctions, even after treatment with CCCP.

Seahorse Xfe 24 Extracellular Flux Analyzer-Based Analysis of Cellular Respiration in Caenorhabditis elegans.

Mitochondria are critical for their role in ATP production as well as multiple nonenergetic functions, and mitochondrial dysfunction is causal in myriad human diseases. Less well appreciated is the fact that mitochondria integrate environmental and intercellular as well as intracellular signals to modulate function. Because mitochondria function in an organismal milieu, there is need for assays capable of rapidly assessing mitochondrial health in vivo. Here, using the Seahorse XF(e) 24 Extracellular Flux Analyzer and the pharmacological inhibitors dicyclohexylcarbodiimide (DCCD, ATP synthase inhibitor), carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP, mitochondrial uncoupler), and sodium azide (cytochrome c oxidase inhibitor), we describe how to obtain in vivo measurements of the fundamental parameters [basal oxygen consumption rate (OCR), ATP-linked respiration, maximal OCR, spare respiratory capacity, and proton leak] of the mitochondrial respiratory chain in the model organism Caenorhabditis elegans.

Quantitative analysis of mitochondrial morphology and membrane potential in living cells using high-content imaging, machine learning, and morphological binning.

Understanding the processes of mitochondrial dynamics (fission, fusion, biogenesis, and mitophagy) has been hampered by the lack of automated, deterministic methods to measure mitochondrial morphology from microscopic images. A method to quantify mitochondrial morphology and function is presented here using a commercially available automated high-content wide-field fluorescent microscopy platform and R programming-language-based semi-automated data analysis to achieve high throughput morphological categorization (puncta, rod, network, and large & round) and quantification of mitochondrial membrane potential. In conjunction with cellular respirometry to measure mitochondrial respiratory capacity, this method detected that increasing concentrations of toxicants known to directly or indirectly affect mitochondria (t-butyl hydroperoxide [TBHP], rotenone, antimycin A, oligomycin, ouabain, and carbonyl cyanide-p-trifluoromethoxyphenylhydrazone [FCCP]), decreased mitochondrial networked areas in cultured 661w cells to 0.60-0.80 at concentrations that inhibited respiratory capacity to 0.20-0.70 (fold change compared to vehicle). Concomitantly, mitochondrial swelling was increased from 1.4- to 2.3-fold of vehicle as indicated by changes in large & round areas in response to TBHP, oligomycin, or ouabain. Finally, the automated identification of mitochondrial location enabled accurate quantification of mitochondrial membrane potential by measuring intramitochondrial tetramethylrhodamine methyl ester (TMRM) fluorescence intensity. Administration of FCCP depolarized and administration of oligomycin hyperpolarized mitochondria, as evidenced by changes in intramitochondrial TMRM fluorescence intensities to 0.33- or 5.25-fold of vehicle control values, respectively. In summary, this high-content imaging method accurately quantified mitochondrial morphology and membrane potential in hundreds of thousands of cells on a per-cell basis, with sufficient throughput for pharmacological or toxicological evaluation.

64Cu-labeled lissamine rhodamine B: a promising PET radiotracer targeting tumor mitochondria.

Enhanced mitochondrial potential in carcinoma cells is an important characteristic of cancer. It is of great current interest to develop a radiotracer that is sensitive to mitochondrial potential changes at the early stage of tumor growth. In this report, we present the synthesis and evaluation of (64)Cu-labeled Lissamine rhodamine B (LRB), (64)Cu(DOTA-LRB) (DOTA-LRB = 2-(6-(diethylamino)-3-(diethyliminio)-3H-xanthen-9-yl)-5-(N-(2-(2-(4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclo-dodecan-1-yl)acetamido)ethyl)sulfamoyl)benzenesulfonate) as a new radiotracer for imaging tumors in athymic nude mice bearing U87MG human glioma xenografts by positron emission tomography (PET). We also explored its localization mechanism using Cu(DOTA-LRB) as the fluorescent probe in both the U87MG human glioma cell line and the cultured primary U87MG glioma cells. It was found that (64)Cu(DOTA-LRB) had the highest tumor uptake (6.54 ± 1.50, 6.91 ± 1.26, 5.68 ± 1.13, 7.58 ± 1.96, and 5.14 ± 1.50%ID/g at 0.5, 1, 2, 4, and 24 h postinjection, respectively) among many (64)Cu-labeled organic cations evaluated in the same animal model. The cellular staining study indicated that Cu(DOTA-LRB) was able to localize in mitochondria of U87MG glioma cells due to the enhanced negative mitochondrial potential. This statement is completely supported by the results from decoupling experiment with carbonylcyanide-m-chlorophenylhydrazone (CCCP). MicroPET data showed that the U87MG glioma tumors were clearly visualized as early as 30 min postinjection with (64)Cu(DOTA-LRB). (64)Cu(DOTA-LRB) remained stable during renal excretion, but underwent extensive degradation during hepatobiliary excretion. On the basis of the results from this study, it was concluded that (64)Cu(DOTA-LRB) represents a new class of promising PET radiotracers for noninvasive imaging of the MDR-negative tumors.

Ca(2+) homeostasis, Ca(2+) signalling and somatodendritic vasopressin release in adult rat supraoptic nucleus neurones.

Multiple mechanisms that maintain Ca(2+) homeostasis and provide for Ca(2+) signalling operate in the somatas and neurohypophysial nerve terminals of supraoptic nucleus (SON) neurones. Here, we examined the Ca(2+) clearance mechanisms of SON neurones from adult rats by monitoring the effects of the selective inhibition of different Ca(2+) homeostatic molecules on cytosolic Ca(2+) ([Ca(2+)](i)) transients in isolated SON neurones. In addition, we measured somatodendritic vasopressin (AVP) release from intact SON tissue in an attempt to correlate it with [Ca(2+)](i) dynamics. When bathing the cells in a Na(+)-free extracellular solution, thapsigargin, cyclopiazonic acid (CPA), carbonyl cyanide 3-chlorophenylhydrazone (CCCP), and the inhibitor of plasma membrane Ca(2+)-ATPase (PMCA), La(3+), all significantly slowed down the recovery of depolarisation (50 mM KCl)-induced [Ca(2+)](i) transients. The release of AVP was stimulated by 50 mM KCl, and the decline in the peptide release was slowed by Ca(2+) transport inhibitors. In contrast to previous reports, our results show that in the fully mature adult rats: (i) all four Ca(2+) homeostatic pathways, the Na(+)/Ca(2+) exchanger, the endoplasmic reticulum Ca(2+) pump, the plasmalemmal Ca(2+) pump and mitochondria, are complementary in actively clearing Ca(2+) from SON neurones; (ii) somatodendritic AVP release closely correlates with intracellular [Ca(2+)](i) dynamics; (iii) there is (are) Ca(2+) clearance mechanism(s) distinct from the four outlined above; and (iv) Ca(2+) homeostatic systems in the somatas of SON neurones differ from those expressed in their terminals.

A high-throughput respirometric assay for mitochondrial biogenesis and toxicity.

Mitochondria are a common target of toxicity for drugs and other chemicals and result in decreased aerobic metabolism and cell death. In contrast, mitochondrial biogenesis restores cell vitality, and there is a need for new agents to induce biogenesis. Current cell-based models of mitochondrial biogenesis or toxicity are inadequate because cultured cell lines are highly glycolytic with minimal aerobic metabolism and altered mitochondrial physiology. In addition, there are no high-throughput real-time assays that assess mitochondrial function. We adapted primary cultures of renal proximal tubular cells (RPTCs) that exhibit in vivo levels of aerobic metabolism, are not glycolytic, and retain higher levels of differentiated functions and used the Seahorse Bioscience analyzer to measure mitochondrial function in real time in multiwell plates. Using uncoupled respiration as a marker of electron transport chain (ETC) integrity, the nephrotoxicants cisplatin, HgCl(2), and gentamicin exhibited mitochondrial toxicity prior to decreases in basal respiration and cell death. Conversely, using FCCP (carbonylcyanide p-trifluoromethoxyphenylhydrazone)-uncoupled respiration as a marker of maximal ETC activity, 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI), SRT1720, resveratrol, daidzein, and metformin produced mitochondrial biogenesis in RPTCs. The merger of the RPTC model and multiwell respirometry results in a single high-throughput assay to measure mitochondrial biogenesis and toxicity and nephrotoxic potential.

Respiration in adipocytes is inhibited by reactive oxygen species.

It is a desirable goal to stimulate fuel oxidation in adipocytes and shift the balance toward less fuel storage and more burning. To understand this regulatory process, respiration was measured in primary rat adipocytes, mitochondria, and fat-fed mice. Maximum O(2) consumption, in vitro, was determined with a chemical uncoupler of oxidative phosphorylation (carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP)). The adenosine triphosphate/adenosine diphosphate (ATP/ADP) ratio was measured by luminescence. Mitochondria were localized by confocal microscopy with MitoTracker Green and their membrane potential (Delta psi(M)) measured using tetramethylrhodamine ethyl ester perchlorate (TMRE). The effect of N-acetylcysteine (NAC) on respiration and body composition in vivo was assessed in mice. Addition of FCCP collapsed Delta psi(M) and decreased the ATP/ADP ratio. However, we demonstrated the same rate of adipocyte O(2) consumption in the absence or presence of fuels and FCCP. Respiration was only stimulated when reactive oxygen species (ROS) were scavenged by pyruvate or NAC: other fuels or fuel combinations had little effect. Importantly, the ROS scavenging role of pyruvate was not affected by rotenone, an inhibitor of mitochondrial complex I. In addition, mice that consumed NAC exhibited increased O(2) consumption and decreased body fat in vivo. These studies suggest for the first time that adipocyte O(2) consumption may be inhibited by ROS, because pyruvate and NAC stimulated respiration. ROS inhibition of O(2) consumption may explain the difficulty to identify effective strategies to increase fat burning in adipocytes. Stimulating fuel oxidation in adipocytes by decreasing ROS may provide a novel means to shift the balance from fuel storage to fuel burning.

Carbonyl cyanide m-chlorophenylhydrazone induced calcium signaling and activation of plasma membrane H(+)-ATPase in the yeast Saccharomyces cerevisiae.

The plasma membrane H(+)-ATPase from Saccharomyces cerevisiae is an enzyme that plays a very important role in the yeast physiology. The addition of protonophores, such as 2,4-dinitrophenol (DNP) and carbonyl cyanide m-chlorophenylhydrazone (CCCP), also triggers a clear in vivo activation of this enzyme. Here, we demonstrate that CCCP-induced activation of the plasma membrane H(+)-ATPase shares some similarities with the sugar-induced activation of the enzyme. Phospholipase C and protein kinase C activities are essential for this activation process while Gpa2p, a G protein involved in the glucose-induced activation of the ATPase, is not required. CCCP also induces a phospholipase C-dependent increase in intracellular calcium. Moreover, we show that the availability of extracellular calcium is required for CCCP stimulation of H(+)-ATPase, suggesting a possible connection between calcium signaling and activation of ATPase.

Calcium regulation in individual peripheral sensory nerve terminals of the rat.

Ca2+ is vital for release of neurotransmitters and trophic factors from peripheral sensory nerve terminals (PSNTs), yet Ca2+ regulation in PSNTs remains unexplored. To elucidate the Ca2+ regulatory mechanisms in PSNTs, we determined the effects of a panel of pharmacological agents on electrically evoked Ca2+ transients in rat corneal nerve terminals (CNTs) in vitro that had been loaded with the fluorescent Ca2+ indicator, Oregon Green 488 BAPTA-1 dextran or fura-2 dextran in vivo. Inhibition of the sarco(endo)plasmic reticulum Ca2+-ATPase, disruption of mitochondrial Ca2+ uptake, or inhibition of the Na+-Ca2+ exchanger did not measurably alter the amplitude or decay kinetics of the electrically evoked Ca2+ transients in CNTs. By contrast, inhibition of the plasma membrane Ca2+-ATPase (PMCA) by increasing the pH slowed the decay of the Ca2+ transient by 2-fold. Surprisingly, the energy for ion transport across the plasma membrane of CNTs is predominantly from glycolysis rather than mitochondrial respiration, as evidenced by the observation that Ca2+ transients were suppressed by iodoacetate but unaffected by mitochondrial inhibitors. These observations indicate that, following electrical activity, the PMCA is the predominant mechanism of Ca2+ clearance from the cytosol of CNTs and glycolysis is the predominant source of energy.

CCCP enhances catecholamine release from the perfused rat adrenal medulla.

The present study was designed to investigate the effect of carbonyl cyanide m-chlorophenylhydrazone (CCCP), a mitochondrial uncoupler, on secretion of catecholamines from the isolated perfused model of the rat adrenal gland and to establish the mechanism of its adrenomedullary secretion. The perfusion of CCCP (3x10(-5) M) into an adrenal vein of for 90 min caused a great increase in catecholamine secretion. Tachyphylaxis to catecholamine-releasing effect of CCCP was not observed by repeated perfusion of it. The net catecholamine-releasing effects of CCCP were depressed by pretreament with pirenzepine (a selective muscarinic M(1)-receptor antagonist), chlorisondamine (a selective neuronal nicotinic receptor antagonist), nicardipine (an L-type Ca2+-channel antagonist), TMB-8 (an intracellular Ca2+-antagonist), and the perfusion of EGTA plus Ca2+-free medium, respectively. In the presence of CCCP (3x10(-5) M), catecholamine secretory responses induced by ACh (5.32x10(-3) M), high K+ (5.6x10(-2) M, a direct membrane depolarizer), DMPP (10(-4) M, (a selective neuronal nicotinic receptor agonist), and McN-A-343 (10(-4) M, (a selective muscarinic M1-receptor agonist) were significantly enhanced. CCCP also significantly enhanced the catecholamine secretory responses evoked by Bay-K-8644 (10(-5) M), L-type Ca2+ channel activator, and cyclopiazonic acid (10(-5) M), an inhibitor of Ca2+-ATPase. Furthermore, the perfusion of FCCP (3x10(-5) M), a similar mitochondrial uncoupler, into an adrenal vein of for 90 min also caused a great increase in catecholamine secretion in a similar pattern with CCCP. Taken together, the results demonstrate that CCCP causes the catecholamine secretion from the perfused rat adrenal medulla in a calcium-dependent fashion. It is thought that this catecholamine secretory enhancement of CCCP may be mediated by both cholinergic receptor stimulation and membrane depolarization, which are relevant to the cytoplasmic Ca2+ increase by stimulation of the Ca2+ influx as well as by the inhibition of Ca2+ uptake into the cytoplasmic Ca2+ stores (both endoplasmic reticulum and mitochondria in chromaffin cells). It also seems that protonophores, such as CCCP, suppress mitochondrial Ca2+ uptake and increase the stimulated secretion of catecholamine by the secretagogues. These results indicate that mitochondria modulate catecholamine secretion by regulating the Ca2+ mobilization for exocytosis.

Cyclopentenone isoprostanes inhibit the inflammatory response in macrophages.

Although both inflammation and oxidative stress contribute to the pathogenesis of many disease states, the interaction between the two is poorly understood. Cyclopentenone isoprostanes (IsoPs), highly reactive structural isomers of the bioactive cyclopentenone prostaglandins PGA2 and PGJ2, are formed non-enzymatically as products of oxidative stress in vivo. We have, for the first time, examined the effects of synthetic 15-A2- and 15-J2-IsoPs, two groups of endogenous cyclopentenone IsoPs, on the inflammatory response in RAW264.7 and primary murine macrophages. Cyclopentenone IsoPs potently inhibited lipopolysaccharide-stimulated IkappaB alpha degradation and subsequent NF-kappaB nuclear translocation and transcriptional activity. Expression of inducible nitric-oxide synthase and cyclooxygenase-2 were also inhibited by cyclopentenone IsoPs as was nitrite and prostaglandin production (IC50 approximately 360 and 210 nM, respectively). 15-J2-IsoPs potently activated peroxisome proliferator-activated receptor gamma (PPARgamma) nuclear receptors, whereas 15-A2-IsoP did not, although the anti-inflammatory effects of both molecules were PPARgamma-independent. Interestingly 15-A2-IsoPs induced oxidative stress in RAW cells that was blocked by the antioxidant 4-hydroxy-TEMPO (TEMPOL) or the mitochondrial uncoupler carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone. TEMPOL also abrogated the inhibitory effect of 15-A2-IsoPs on lipopolysaccharide-induced NF-kappaB activation, inducible nitricoxide synthase expression, and nitrite production, suggesting that 15-A2-IsoPs inhibit the NF-kappaB pathway at least partially via a redox-dependent mechanism. 15-J2-IsoP, but not 15-A2-IsoP, also potently induced RAW cell apoptosis again via a PPAR gamma-independent mechanism. These findings suggest that cyclopentenone IsoPs may serve as negative feedback regulators of inflammation and have important implications for defining the role of oxidative stress in the inflammatory response.

Reactive oxygen species regulate caspase activation in tumor necrosis factor-related apoptosis-inducing ligand-resistant human colon carcinoma cell lines.

The effects of reactive oxygen species (ROS) on tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in solid cancers have yet to be clearly defined. In this study, we found that the classic uncoupler of oxidative phosphorylation, carbonyl cyanide m-chlorophenylhydrazone (CCCP), induced a reduction in DeltaPsim and generation of ROS. This uncoupling effect enhanced TRAIL-induced apoptosis in TRAIL-resistant human colon carcinoma cell lines (RKO, HT29, and HCT8). Sensitization was inhibited by benzyloxycarbonyl-valine-alanine-aspartate fluoromethylketone, indicating the requirement for caspase activation. CCCP per se did not induce apoptosis or release of proapoptotic factors from mitochondria. Generation of ROS by CCCP was responsible for TRAIL-induced Bax and caspase activation because scavenging ROS completely abrogated apical caspase-8 activation and further downstream events leading to cell death. Overexpression of Bcl-2 did not prevent the initial loss of DeltaPsim and ROS generation following CCCP treatment, but did prevent cell death following TRAIL and CCCP exposure. Uncoupling of mitochondria also facilitated TRAIL-induced release of proapoptotic factors. X-linked inhibitor of apoptosis overexpression abrogated TRAIL-induced apoptosis in the presence of CCCP and decreased initiator procaspase-8 processing, indicating that additional processing of caspase-8 required initiation of a mitochondrial amplification loop via effector caspases. Of interest, depletion of caspase-9 in RKO cells did not protect cells from TRAIL/CCCP-induced apoptosis, indicating that apoptosis occurred via a caspase-9-independent pathway. Data suggest that in the presence of mitochondrial-derived ROS, TRAIL induced mitochondrial release of Smac/DIABLO and inactivation of X-linked inhibitor of apoptosis through caspase-9-independent activation of caspase 3.

Mitochondria-derived reactive oxygen species dilate cerebral arteries by activating Ca2+ sparks.

Mitochondria regulate intracellular calcium (Ca2+) signals in smooth muscle cells, but mechanisms mediating these effects, and the functional relevance, are poorly understood. Similarly, antihypertensive ATP-sensitive potassium (KATP) channel openers (KCOs) activate plasma membrane KATP channels and depolarize mitochondria in several cell types, but the contribution of each of these mechanisms to vasodilation is unclear. Here, we show that cerebral artery smooth muscle cell mitochondria are most effectively depolarized by diazoxide (-15%, tetramethylrhodamine [TMRM]), less so by levcromakalim, and not depolarized by pinacidil. KCO-induced mitochondrial depolarization increased the generation of mitochondria-derived reactive oxygen species (ROS) that stimulated Ca2+ sparks and large-conductance Ca2+-activated potassium (KCa) channels, leading to transient KCa current activation. KCO-induced mitochondrial depolarization and transient KCa current activation were attenuated by 5-HD and glibenclamide, KATP channel blockers. MnTMPyP, an antioxidant, and Ca2+ spark and KCa channel blockers reduced diazoxide-induced vasodilations by >60%, but did not alter dilations induced by pinacidil, which did not elevate ROS. Data suggest diazoxide drives ROS generation by inducing a small mitochondrial depolarization, because nanomolar CCCP, a protonophore, similarly depolarized mitochondria, elevated ROS, and activated transient KCa currents. In contrast, micromolar CCCP, or rotenone, an electron transport chain blocker, induced a large mitochondrial depolarization (-84%, TMRM), reduced ROS, and inhibited transient KCa currents. In summary, data demonstrate that mitochondria-derived ROS dilate cerebral arteries by activating Ca2+ sparks, that some antihypertensive KCOs dilate by stimulating this pathway, and that small and large mitochondrial depolarizations lead to differential regulation of ROS and Ca2+ sparks.

Ca2+ clearance mechanisms in neurohypophysial terminals of the rat.

The importance of intracellular calcium ([Ca2+]i) in the release of vasopressin (AVP) and oxytocin from the central nervous system neurohypopyhysial nerve terminals has been well-documented. To date, there is no clear understanding of Ca2+ clearance mechanisms and their interplay with transmembrane Ca2+ entry, intracellular [Ca2+]i transients, cytoplasmic Ca2+ stores and hence the release of AVP at the level of a single nerve terminal. Here, we studied the mechanism of Ca2+ clearance in freshly isolated nerve terminals of the rat neurohypophysis using Fura-2 Ca2+ imaging and measured the release of AVP by radioimmuno assay. An increase in the K+ concentration in the perfusion solution from 5 to 50 mM caused a rapid increase in [Ca2+]i and AVP release. Returning K+ concentration to 5 mM led to rapid restoration of both responses to basal level. The K+-evoked [Ca2+]i and AVP increase was concentration-dependent, reliable, and remained of constant amplitude and time course upon successive applications. Extracellular Ca2+ removal completely abolished the K+-evoked responses. The recovery phase was not affected upon replacement of NaCl with sucrose or drugs known to act on intracellular Ca2+ stores such as thapsigargin, cyclopiazonic acid, caffeine or a combination of caffeine and ryanodine did not affect either resting or K+-evoked [Ca2+]i or AVP release. By contrast, the plasma membrane Ca2+ pump inhibitor, La3+, markedly slowed down the recovery phase. The mitochondrial respiration uncoupler, carbonyl cyanide 3-chlorophenylhydrazone (CCCP), slightly but significantly increased the basal [Ca2+]i, and also slowed down the recovery phase of both [Ca2+]i and release responses. In conclusion, we show in nerve terminals that (i) Ca2+ extrusion through the Ca2+ pump in the plasma membrane plays a major role in the Ca2+ clearance mechanisms of (ii) Ca2+ uptake by mitochondria also contributes to the Ca2+ clearance and (iii) neither Na+/Ca2+ exchangers nor Ca2+ stores are involved in the Ca2+ clearance or in the maintenance of basal [Ca2+]i or release of AVP.

[Kinetic model of spermine effect on the Mg2+, ATP-dependent transport of Ca ions in the smooth muscle mitochondria].

In experiments, carried out with the use of a radioactive label (45Ca2+) on suspension of rat uterus myocytes treated with digitonin solution (0.1 mg/ml), influence of spermine on the Mg2+, ATP-dependent Ca2+ transport in the mitochondria was investigated. Ca2+ accumulation in the mitochondria was tested as such which was blocked by ruthenium red (10 microM) and was not sensitive to thapsigargin (100 nM). It was shown, that dependence of initial speed of Ca ions accumulation in the mitochondria on spermine concentration (0.1-10 mm) is described by a bell-shaped curve. Spermine concentration being increased in the range of 0.1-1 mM the stimulation of Ca2+ accumulation was observed, at the further increase in polyamine concentration up to 10 mM the suppression of this process took place. On the basis of the analysis of the authors' experimental results and the literature data the model of complex spermine action on Ca2+ accumulation in mitochondria was proposed and analyzed. The existence of two spermine binding sites on mitochondrial membrane--S1 and S2 occupation of which is connected to activation and inhibition of Ca(2+)-unipoter, accordingly, was taken into account. The kinetic analysis of the model which has been made in an equilibrium mode, allowed to calculate some important quantitative parameters describing spermine influence on Ca ions accumulation in mitochondria. It is supposed, that the proposed model can be useful in the further research of polyamine influence on transmembrane exchange of Ca ions in mitochondria.