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Absorbance spectroscopy of intrinsic cardiac chromophores provides nondestructive assessment of cytosolic oxygenation and mitochondria redox state. Isolated perfused heart spectroscopy is usually conducted by collecting reflected light from the heart surface, which represents a combination of surface scattering events and light that traversed portions of the myocardium. Reflectance spectroscopy with complex surface scattering effects in the beating heart leads to difficulty in quantitating chromophore absorbance. In this study, surface scattering was minimized and transmural path length optimized by placing a light source within the left ventricular chamber while monitoring transmurally transmitted light at the epicardial surface. The customdesigned intrachamber light catheter was a flexible coaxial cable (2.42-Fr) terminated with an encapsulated side-firing LED of 1.8 x 0.8 mm, altogether similar in size to a Millar pressure catheter. The LED catheter had minimal impact on aortic flow and heart rate in Langendorff perfusion and did not impact stability of the left ventricule of the working heart. Changes in transmural absorbance spectra were deconvoluted using a library of chromophore reference spectra to quantify the relative contribution of specific chromophores to the changes in measured absorbance. This broad-band spectral deconvolution approach eliminated errors that may result from simple dualwavelength absorbance intensity. The myoglobin oxygenation level was only 82.2 ± 3.0%, whereas cytochrome c and cytochrome a + a3 were 13.3 ± 1.4% and 12.6 ± 2.2% reduced, respectively, in the Langendorff-perfused heart. The intracardiac illumination strategy permits transmural optical absorbance spectroscopy in perfused hearts, which provides a noninvasive real-time monitor of cytosolic oxygenation and mitochondria redox state. [ABSTRACT FROM AUTHOR]

Divalent metal-ion transporter-1 (DMT1), the principal mechanism by which nonheme iron is taken up at the intestinal brush border, is energized by the H+-electrochemical potential gradient. The provenance of the H+ gradient in vivo is unknown, so we have explored a role for brush-border Na+/H+ exchanger (NHE) isoforms by examining iron homeostasis and intestinal iron handling in mice lacking NHE2 or NHE3. We observed modestly depleted liver iron stores in NHE2-null (NHE2-/-) mice stressed on a low-iron diet but no change in hematological or blood iron variables or the expression of genes associated with iron metabolism compared with wild-type mice. Ablation of NHE3 strongly depleted liver iron stores, regardless of diet. We observed decreases in blood iron variables but no overt anemia in NHE3-null (NHE3-/-) mice on a low-iron diet. Intestinal expression of DMT1, the apical surface ferrireductase cytochrome b reductase-1, and the basolateral iron exporter ferroportin was upregulated in NHE3-/- mice, and expression of liver Hamp1 (hepcidin) was suppressed compared with wild-type mice. Absorption of 59Fe from an oral dose was substantially impaired in NHE3-/- compared with wild-type mice. Our data point to an important role for NHE3 in generating the H+ gradient that drives DMT1-mediated iron uptake at the intestinal brush border. [ABSTRACT FROM AUTHOR]

Resting metabolic rate (RMR) in humans shows pronounced individual variations, but the underlying molecular mechanism remains elusive. Cytochrome c oxidase (COX) plays a key role in control of metabolic rate, and recent studies of the subunit 4 isoform 2 (COX IV-2) indicate involvement in the cellular response to hypoxia and oxidative stress. We evaluated whether the COX subunit IV isoform composition may explain the pronounced individual variations in resting metabolic rate (RMR). RMR was determined in healthy humans by indirect calorimetry and correlated to levels of COX IV-2 and COX IV-1 in vastus lateralis. Overexpression and knock down of the COX IV isoforms were performed in primary myotubes followed by evaluation of the cell respiration and production of reactive oxygen species. Here we show that COX IV-2 protein is constitutively expressed in human skeletal muscle and strongly correlated to RMR. Primary human myotubes overexpressing COX IV-2 displayed markedly (>60%) lower respiration, reduced (>50%) cellular H2O2 production, higher resistance toward both oxidative stress, and severe hypoxia compared with control cells. These results suggest an important role of isoform COX IV-2 in the control of energy expenditure, hypoxic tolerance, and mitochondrial ROS homeostasis in humans. [ABSTRACT FROM AUTHOR]

ategorization as a cytochrome P-450 (CYP) 2C19 poor metabolizer (PM) is reported to be an independent risk factor for cardiovascular disease. It is correlated with an increase in the circulating levels of high-sense C-reactive protein (hs-CRP) in women only, although its role in coronary microcirculation is unclear. We examined sex differences in the impact of the CYP2C19 genotype and low-grade inflammation on coronary microvascular disorder (CMVD). We examined CYP2C19 genotypes in patients with CMVD (n=81) and in healthy subjects as control (n=81). CMVD was defined as the absence of coronary artery stenosis and epicardial spasms, the presence of inverted lactic acid levels between the intracoronary and coronary sinuses, or an adenosine triphosphate-induced coronary flow reserve ratio < 2.5. CYP2C19 PMs have two loss-of-function (LOF) alleles (*2, *3). Extensive metabolizers have no LOF alleles, and intermediate metabolizers have one LOF allele. The ratio of CYP2C19 PM and hs-CRP levels in CMVD was significantly higher than that of controls, especially in women (40.9 vs. 13.8%, P = 0.013; 0.11 ± 0.06 vs. 0.07 ± 0.04 mg/dl, P = 0.001). Moreover, in each CYP2C19 genotype, hs-CRP levels in CMVD in CYP2C19 PMs were significantly higher than those of the controls, especially in women (0.15 ± 0.06 vs. 0.07 ± 0.03, P = 0.004). Multivariate analysis for CMVD indicated that the female sex, current smoking, and hypertension were predictive factors, and that high levels of hs-CRP and CYP2C19 PM were predictive factors in women only (odds ratio 3.5, 95% confidence interval 1.26-9.93, P = 0.033; odds ratio 4.1, 95% confidence interval 1.15-14.1, P = 0.038). CYP2C19 PM genotype may be a new candidate risk factor for CMVD via inflammation exclusively in the female population. [ABSTRACT FROM AUTHOR]

Mitochondrial control of cellular redox states is a fundamental component of cell signaling in the coordination of core energy metabolism and homeostasis during normoxia and hypoxia. We investigated the relationship between cytochrome redox states and mitochondrial oxygen consumption at steady-state levels of hypoxia in mitochondria isolated from beef and mouse heart (BHImt, MHImt), comparing two species with different cardiac dynamics and local oxygen demands. A low-noise, rapid spectrophotometric system using visible light for the measurement of cytochrome redox states was combined with high-resolution respirometry. Monophasic hyperbolic relationships were observed between oxygen consumption, JO2, and oxygen partial pressure, PO2, within the range <1.1 kPa (8.3 mmHg; 13 μM). P50j (PO2 at 0.5.Jmax) was 0.015 ± 0.0004 and 0.021 ± 0.003 kPa (0.11 and 0.16 mmHg) for BHImt and MHImt, respectively. Maximum oxygen consumption, Jmax, was measured at saturating ADP levels (OXPHOS capacity) with Complex I-linked substrate supply. Redox states of cytochromes aa3 and c were biphasic hyperbolic functions of PO2. The relationship between cytochrome oxidation state and oxygen consumption revealed a separation of distinct phases from mild to severe and deep hypoxia. When cytochrome c oxidation increased from fully reduced to 45% oxidized at 0.1 Jmax, PO2 was as low as 0.002 kPa (0.02 μM), and trace amounts of oxygen are sufficient to partially oxidize the cytochromes. At higher PO2 under severe hypoxia, respiration increases steeply, whereas redox changes are small. Under mild hypoxia, the steep slope of oxidation of cytochrome c when flux remains more stable represents a cushioning mechanism that helps to maintain respiration high at the onset of hypoxia. [ABSTRACT FROM AUTHOR]

Mitochondrial oxidative phosphorylation has a central role in eukaryotic metabolism, providing the energy (ATP) required for survival. Regulation of this important pathway is, however, still not understood, largely due to limitations in the ability to measure the essential metabolites, including oxygen (pO2, oxygen pressure), ADP, and AMP. In addition, neither the mechanism of oxygen reduction by mitochondrial cytochrome c oxidase nor how its rate is controlled is understood, although this enzyme determines the rate of oxygen consumption and thereby the rate of ATP synthesis. Cytochrome c oxidase is responsible for reduction of molecular oxygen to water using reducing equivalents donated by cytochrome c and for site 3 energy coupling in oxidative phosphorylation. A mechanism-based model of the cytochrome c oxidase reaction is presented in which transfer of reducing equivalents from the lower-to the higher-potential region of the coupling site occurs against an opposing energy barrier, Q. The steady-state rate equation is fitted to data for the dependence of mitochondrial respiratory rate on cytochrome c reduction, oxygen pressure (pO2), and [ATP]/[ADP][Pi] atpH 6.5 to 8.35 (where Pi is inorganic phosphate). The fit of the rate expression to the experimental data is very good for all experimental conditions. Levels of the intermediates in oxygen reduction in the oxidase reaction site have been calculated. An intermediate in the reaction, tentatively identified as peroxide, bridged between the iron and copper atoms of the reaction site has a central role in coupling mitochondrial respiration to the [ATP]/[ADP][Pi]. [ABSTRACT FROM AUTHOR]

Cytochrome c oxidase is the enzyme responsible for oxygen consumption by mitochondrial oxidative phosphorylation and coupling site 3 of oxidative phosphorylation. In this role it determines the cellular rate of ATP synthesis by oxidative phosphorylation and is the key to understanding how energy metabolism is regulated. Four electrons are required for the reduction of oxygen to water, and these are provided by the one-electron donor, cytochrome c. The rate of oxygen consumption (ATP synthesis) is dependent on the fraction of cytochrome c reduced (fred), oxygen pressure (pO2), energy state ([ATP]/[ADP][Pi]), and pH. In coupled mitochondria (high energy state) and pO2 >60 torr, the rate increases in an exponential-like fashion with increasing fred. When the dependence on fred is fitted to the equation rate = a(fred)b, a decreased from 100 to near 20, and b increased from 1.3 to 4 as the pH of the medium increased from 6.5 to 8.3. During oxygen depletion from the medium fred progressively increases and the rate of respiration decreases. The respiratory rate falls to ½ (P50) by about 1.5 torr, at which point fred is substantially increased. The metabolically relevant dependence on pO2 is obtained by correcting for the increase in fred, in which case the P50 is 12 torr. Adding an uncoupler of oxidative phosphorylation eliminates the dependence of the cytochrome c oxidase activity on pH and energy state. The respiratory rate becomes proportional to fred and the P50 decreases to less than 1 torr. [ABSTRACT FROM AUTHOR]

Nephrotoxicity is one of the important dose-limiting factors during cisplatin treatment. There is a growing body of evidence that activation of p53 has a critical role in cisplatin-induced renal apoptotic injury. The nicotinamide adenine dinucleotide-dependent protein deacetylase SIRT1 decreases apoptosis through deacetylating of p53, and resveratrol is known as an activator of SIRT1. To study the role of SIRT1 in cisplatin-induced renal injury through interaction with p53, mouse proximal tubular cells (MPT) were treated with cisplatin and examined the expression level of SIRT1, acetylation of p53, PUMA-α, Bax, the cytosolic/mitochondrial cytochrome c ratio, and active caspase-3. The expression of SIRT1 was decreased by cisplatin. Resveratrol, a SIRT1 activator, ameliorated cisplatin-induced acetylation of p53, apoptosis, and cytotoxicity in MPT cells. In addition, resveratrol remarkably blocked cisplatin-induced decrease of Bcl-xL in MPT cells. Further specific SIRT1 inhibition with EX 527 or small interference RNA specific to SIRT1 reversed the effect of resveratrol on cisplatin-induced toxicity. Inhibition of p53 by pifithrin-α reversed the effect of EX527 in protein expression of PUMA-α, Bcl-xL, and caspase-3 and cytotoxicity in MPT cells. SIRT1 protein expression after cisplatin treatment was significantly decreased in the kidney. SIRT1 activation by resveratrol decreased cisplatin-induced apoptosis while improving the glomerular filtration rate. Taken together, our findings suggest that the modulation of p53 by SIRT1 could be a possible target to attenuate cisplatin-induced kidney injury. [ABSTRACT FROM AUTHOR]

Alterations in renal function contribute to Goldblatt two-kidney, one-clip (2K1C) hypertension. A previous study indicated that bioavailability of cytochrome P-450 metabolites epoxyeicosatrienoic acids (EETs) is decreased while that of 20-hydroxyeicosatetraenoic acids (20-HETE) is increased in this model. We utilized the inhibitor of soluble epoxide hydrolase cis-4-[4-(3-adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid (c-AUCB) and HET-0016, the inhibitor of 20-HETE production, to study the role of EETs and 20-HETE in the regulation of renal function. Chronic c-AUCB treatment significantly decreased systolic blood pressure (SBP) (133 ± 1 vs. 163 ± 3 mmHg) and increased sodium excretion (1.23 ± 0.10 vs. 0.59 ± 0.03 mmol/day) in 2K1C rats. HET-0016 did not affect SBP and sodium excretion. In acute experiments, renal blood flow (RBF) was decreased in 2K1C rats (5.0 ± 0.2 vs. 6.9 ± 0.2 ml∙min-1∙g-1). c-AUCB normalized RBF in 2K1C rats (6.5 ± 0.6 ml∙min-1∙g-1). HET-0016 also increased RBF in 2K1C rats (5.8 ± 0.2 ml∙min-1∙g-1). Although RBF and glomerular filtration rate (GFR) remained stable in normotensive rats during renal arterial pressure (RAP) reductions, both were significantly reduced at 100 mmHg RAP in 2K1C rats. c-AUCB did not improve autoregulation but increased RBF at all RAPs and shifted the pressure-natriuresis curve to the left. HET-0016-treated 2K1C rats exhibited impaired autoregulation of RBF and GFR. Our data indicate that c-AUCB displays antihypertensive properties in 2K1C hypertension that are mediated by an improvement of RBF and pressure natriuresis. While HET-0016 enhanced RBF, its anti-natriuretic effect likely prevented it from producing a blood pressure-lowering effect in the 2K1C model. [ABSTRACT FROM AUTHOR]

While myogenic force in response to a changing arterial pressure has been described early in the 20th century, it was not until 1984 that the effect of a sequential increase in intraluminal pressure on cannulated cerebral arterial preparations was found to result in pressure-dependent membrane depolarization associated with spike generation and reduction in lumen diameter. Despite a great deal of effort by different laboratories and investigators, the identification of the existence of a mediator of the pressure-induced myogenic constriction in arterial muscle remained a challenge. It was the original finding by our laboratory that demonstrated the capacity of cerebral arterial muscle cells to express the cytochrome P-450 4A enzyme that catalyzes the formation of the potent vasoconstrictor 20-hydroxyeicosatetraenoic acid (20-HETE) from arachidonic acid, the production of which in cerebral arterial muscle cells increases with the elevation in intravascular pressure. 20-HETE activates protein kinase C and causes the inhibition of Ca2+-activated K+ channels, depolarizes arterial muscle cell membrane, and activates L-type Ca2+ channel to increase intracellular Ca2+ levels and evoke vasoconstriction. The inhibition of 20-HETE formation attenuates pressure-induced arterial myogenic constriction in vitro and blunts the autoregulation of cerebral blood flow in vivo. We suggest that the formation and action of cytochrome P-450-derived 20-HETE in cerebral arterial muscle could play a critically important role in the control of cerebral arterial tone and the autoregulation of cerebral blood flow under physiological conditions. [ABSTRACT FROM AUTHOR]

Skeletal muscle contractions increase superoxide anion in skeletal muscle extracellular space. We tested the hypotheses that 1) after an isometric contraction protocol, xanthine oxidase (XO) activity is a source of superoxide anion in the extracellular space of skeletal muscle and 2) the increase in XO-derived extracellular superoxide anion during contractions affects skeletal muscle contractile function. Superoxide anion was monitored in the extracellular space of mouse gastrocnemius muscles by following the reduction of cytochrome cm muscle microdialysates. A 15-mm protocol of nondamaging isometric contractions increased the reduction of cytochrome c in microdialysates, indicating an increase in superoxide anion. Mice treated with the XO inhibitor oxypurinol showed a smaller increase in superoxide anions in muscle microdialysates following contractions than in microdialysates from muscles of vehicle-treated mice. Intact extensor digitorum longus (EDL) and soleus muscles from mice were also incubated in vitro with oxypurinol or polyethylene glycol-tagged Cu,Zn-SOD. Oxypurinol decreased the maximum tetanic force produced by EDL and soleus muscles, and polyethylene glycol-tagged Cu,Zn-SOD decreased the maximum force production by the EDL muscles. Neither agent influenced the rate of decline in force production when EDL or soleus muscles were repeatedly electrically stimulated using a 5-mm fatiguing protocol (stimulation at 40 Hz for 0.1 s every 5 s). Thus these studies indicate that XO activity contributes to the increased superoxide anion detected within the extracellular space of skeletal muscles during nondamaging contractile activity and that XO-derived superoxide anion or derivatives of this radical have a positive effect on muscle force generation during isometric contractions of mouse skeletal muscles. [ABSTRACT FROM AUTHOR]

It is proposed that ischemic preconditioning (PC) initiates signaling that converges on mitochondria and results in cardioprotection. The outcome of this signaling on mitochondrial enzyme complexes is yet to be understood. We therefore used proteomic methods to test the hypothesis that PC and pharmacological preconditioning similarly alter mitochondrial signaling complexes. Langendorff-perfused murine hearts were treated with the specific GSK-3 inhibitor AR-A014418 (GSK Inhib VIII) for 10 mm or subjected to four cycles of 5-mm ischemiareperfusion (PC) before 20-mm global ischemia and 120-mm reperfusion. PC and OSK Inhib VIII both improved recovery of postischemic left ventricular developed pressure, decreased infarct size, and reduced lactate production during ischemia compared with their time-matched controls. We used proteomics to examine mitochondrial protein levels/posttranslational modifications that were common between PC and OSK Inhib VIII. Levels of cytochrome-c oxidase subunits Va and Vib, ATP synthase-coupling factor 6, and cytochrome b-cl complex subunit 6 were increased while cytochrome c was decreased with PC and GSK Inhib VIII. Furthermore, the amount of cytochrome-c oxidase subunit VIb was found to be increased in PC and GSK Inhib VIII mitochondrial supercomplexes, which are comprised of complexes I, III, and IV. This result would suggest that changes in complex subunits associated with cardioprotection may affect supercomplex composition. Thus the ability of PC and GSK inhibition to alter the expression levels of electron transport complexes will have important implications for mitochondrial function. [ABSTRACT FROM AUTHOR]

Rottlerin is a polyphenolic compound derived from Mallotus philipinensis. In the present study, we show that rottlerin decreased tumor size and stimulated apoptosis in an orthotopic model of pancreatic cancer with no effect on normal tissues in vivo. Rottlerin also induced apoptosis in pancreatic cancer (PaCa) cell lines by interacting with mitochondria and stimulating cytochrome c release. Immunoprecipitation results indicated that rottlerin disrupts complexes of prosurvival Bcl-xL with Bim and Puma. Furthermore, siRNA knockdown showed that Bim and Puma are necessary for rottlerin to stimulate apoptosis. We also showed that rottlerin and Bcl-2 and Bcl-xL inhibitor BH3I-2' stimulate apoptosis through a common mechanism. They both directly interact with mitochondria, causing increased cytochrome c release and mitochondrial depolarization, and both decrease sequestration of BH3-only proteins by Bcl-xL. However, the effects of rottlerin and BH3I-2' on the complex formation between Bcl-xL and BH3-only proteins are different. BH3I-2' disrupts complexes of Bcl-xL with Bad but not with Bim or Puma, whereas rottlerin had no effect on the Bcl-xL interaction with Bad. Also BH3I-2', but not rottlerin, required Bad to stimulate apoptosis. In conclusion, our results demonstrate that rottlerin has a potent proapoptotic and antitumor activity in pancreatic cancer, which is mediated by disrupting the interaction between prosurvival Bcl-2 proteins and proapoptotic BH3-only proteins. Thus rottlerin represents a promising novel agent for pancreatic cancer treatment. [ABSTRACT FROM AUTHOR]

Liu K, Zhou S, Kim J, Tillison K, Majors D, Rearick D, Lee .JH, FernanJez-Boyanapalli RF, Barricklow K, Houston MS, Smas CM. Functional analysis of FSP27 protein regions for lipid droplet localization, caspase-dependent apoptosis, and dimerization with CIDEA. Am J Physiol Endocrinol Metab 297: El 395-E14 13, 2009. First published October 20, 2009; doi:10.1152/ajpendo.00188.2009.— The adipocyte-specific protein FSP27, also known as CIDEC, is one of three cell death-inducing DFF45-like effector (CIDE) proteins. The first known function for CIDEs was promotion of apoptosis upon ectopic expression in mammalian cells. Recent studies in endogenous settings demonstrated key roles for CIDEs in energy metabolism. FSP27 is a lipid dioplet-associated protein whose heterologous expression enhances formation of enlarged lipid droplets and is required for unilocular lipid droplets typical of white adipocytes in vivo. Here, we delineate relationships between apoptotic function and lipid droplet localization of FSP27. We demonstrate that ectopic expression of FSP27 induces enlarged lipid droplets in multiple human cell lines, which is indicative that its mechanism involves ubiquitously present, rather than adipocyte-specific, cellular machinery. Furthermore, promotion of lipid droplet formation in HeLa cells via culture in exogenous oleic acid offsets FSP27-mediated apoptosis. Using transient cotransfections and analysis of lipid droplets in HeLa cells stably expressing FSP27, we show that FSP27 does not protect lipid droplets from action of ATGL lipase. Domain mapping with eGFP-FSP27 deletion constructs indicates that lipid droplet localization of FSP27 require:; amino acids 174-192 of its CIDE C domain. The apoptotic mecharism of FSP27, which we show involves caspase-9 and mitochondrial cytochrome c, also requires this 19-amino acid region. Interaction assays determine the FSP27 CIDE C domain complexes with CIDEA, and Western blot reveals that FSP27 protein levels are reduced by coexpression of CIDEA. Overall, our findings demonstrate the function of the FSP27 CIDE C domain and/or regions thereof for apoptosis, lipid droplet localization, and CIDEA interaction. [ABSTRACT FROM AUTHOR]

Nephrotoxicity is the major limiting factor for the use of cisplatin in cancer therapy. Recent studies have demonstrated an important role for p53 in cisplatin-induced renal injury. Nevertheless, pharmacological and genetic blockade of p53 only provides partial renoprotective effects, suggesting the presence of p53-independent injury mechanisms. To understand the p53-independent mechanisms, we have now examined cisplatin-induced apoptosis in p53-deficient kidney cells. We show that cisplatin could induce Bax activation, cytochrome c release, and apoptosis in primary cultures of p53-deficient renal tubular cells, albeit at a level that was lower than in the wild-type cells. Cisplatin could also induce typical apoptosis in p53-deficient baby mouse kidney (BMK) cells. The apoptosis was caspase dependent and could be completely blocked by general caspase inhibitors. Bax and Bak, two key molecules in the mitochondrial pathway of apoptosis, were interdependently activated by cisplatin, with Bax translocation to and Bax/Bak oligomerization in mitochondria, leading to cytochrome c release. Importantly, cytochrome c release and apoptosis were diminished in Bax/Bak single or double-knockout BMK cells. Furthermore, overexpression of Bcl-2 could ameliorate cisplatin-induced cytochrome c release and apoptosis. Together, the results have demonstrated a p53-independent mechanism of cisplatin nephrotoxicity that involves the mitochondrial pathway of apoptosis. [ABSTRACT FROM AUTHOR]

Ferré N, MartInez-Clemente M, López-Parra M, GonzalezPériz A, Horrillo R, Planagumà A, Camps J, Joven J, Tres A, Guardiola F, Batalier R, Arroyo V, Clària J. Increased susceptibility to exacerbated liver injury in hypercholesterolemic ApoEdeficient mice: potential involvement of oxysterols. Am J Physiol Gastrointest Liver Physiol 296: G553-G562, 2009. First published January 8, 2009; doi:10.1152/ajpgi.00547.2007.-The contribution of metabolic factors to the severity of liver disease is not completely understood. In this study, apolipoprotein E-deficient (ApoE) mice were evaluated to define potential effects of hypercholesterolemia on the severity of carbon tetrachloride (CC1[sub4])-induced liver injury. Under baseline conditions, hypercholesterolemic ApoE[sup-/-] mice showed increased hepatic oxidative stress (SOD activity/4-hydroxy-2-nonenal immunostaining) and higher hepatic TGF-β[sub1], MCP-1, and TIMP-l expression than wild-type control mice. After CC1[sub4] challenge, ApoE[sup-/-] mice exhibited exacerbated steatosis (Oil Red 0 staining), necroinflammation (hematoxylin-eosin staining), macrophage infiltration (F4/80 immunohistochemistly), and fibrosis (Sirius red staining and a-smooth muscle actin immunohistochemistry) and more severe liver injury [alanine aminotransferase (ALT) and aspartate aminotransferase] than wild-type controls. Direct conelations were identified between serum cholesterol and hepatic steatosis, fibrosis, and ALT levels. These changes did not reflect the usual progression of the disease in ApoE[sup-/-] mice, since exacerbated liver injury was not present in untreated age-paired ApoE[sup-/-] mice. Moreover, hepatic cytochrome P-450 expression was unchanged in ApoE[sup-/-] mice. To explore potential mechanisms, cell types relevant to liver pathophysiology were exposed to selected cholesterol-oxidized products. Incubation of hepatocytes with a mixture of oxysterols representative of those detected by GC-MS in livers from ApoE[sup-/-] mice resulted in a concentration-dependent increase in total lipoperoxides and SOD activity. In hepatic stellate cells, oxysterols increased IL-8 secretion through a NF-KB-independent mechanism and upregulated TIMP-l expression. In macrophages, oxysterols increased TGF-β[sub1] secretion and MCP-1 expression in a concentration-dependent manner. Oxysterols did not compromise cell viability. Taken together, these findings demonstrate that hypercholesterolemic mice are sensitized to liver injury and that cholesterol-derived products (i.e., oxysterols) are able to induce promflanimatory and profibrogenic mechanisms in liver cells. [ABSTRACT FROM AUTHOR]

Physical exercise training is a powerful tool to maintain or improve mitochondrial density and function (mitochondrial capacity). This study aims to determine whether mitochondrial capacity is also associated with habitual physical activity in daily life (PADL). The capacity of classic markers for mitochondrial density, i.e., the capacity of citrate synthase (CS) and succinate dehydrogenase (SDH), as well the capacity of cytochrome c oxidase (COX) and β-hydroxyacyl-CoA dehydrogenase (HAD), was determined in homogenized muscle biopsy samples obtained from the vastus lateralis muscle of nonexercising healthy young (age 20 ± 2 yr) subjects (31 women, 7 men). PADL was measured during two periods of 14 days using a triaxial accelerometer for movement registration. CS, SDH, and COX were positively associated with PADL [P < 0.05, R 0.36, 95% confidence interval (CI): 1.3·10-5 to 2.2·10-3 P < 0.05, R = 0.39, 95% CI: 1.1·10-5 to 9.9·10-5 and P < 0.05, R = 0.33, 95% CI: 7.5·10-6 to 3.6·10-3, respectively], and HAD tended to correlate positively with PADL (P = 0.06, R = 0.31, 95% CL: -2.2·10-5 to 1.1·10-3). The population was subsequently stratified based on the intensity of the activities performed. CS was only associated with PADL in subjects spending more time on high-intensity physical activity, whereas HAD was only associated with PADL in subjects spending less time on low intensity physical activity. We are the first to report that even within the range of normal daily life activities, mitochondrial capacity is positively associated with the level of habitual physical activity in daily life. Thus an active lifestyle may help to maintain or improve mitochondrial capacity. [ABSTRACT FROM AUTHOR]

During low O2 (hypoxia), hypoxia-inducible factor (HIF)-α is stabilized and translocates to the nucleus, where it regulates genes critical for survival and/or adaptation in low O2. While it appears that mitochondria play a critical role in HIF induction, controversy surrounds the underlying mechanism(s). To address this, we monitored HIF-2α expression and oxygen consumption in an O2-sensitive immortalized rat adrenomedullary chromaffin (MAH) cell line. Hypoxia (2-8% O2) caused a concentration- and time-dependent increase in HIF-2α induction, which was blocked in MAH cells with either RNA interference knockdown of the Rieske Fe-S protein, a component of complex III, or knockdown of cytochrome-c oxidase subunit of complex IV, or defective mitochondrial DNA (p0 cells). Additionally, pharmacological inhibitors of mitochondrial complexes I, III, IV, i.e., rotenone (1 p~M), myxothiazol (1 μM), antimycin A (1 μg/ml), and cyanide (1 mM), blocked HIF-2α induction in control MAH cells. Interestingly, the inhibitory effects of the mitochondrial inhibitors were dependent on O2 concentration such that at moderate-to-severe hypoxia (6% O2), HIF-2α induction was blocked by low inhibitor concentrations that were ineffective at more severe hypoxia (2% O2). Manipulation of the levels of reactive oxygen species (ROS) had no effect on HIF-2α induction. These data suggest that in this O2-sensitive cell line, mitochondrial 02 consumption, rather than changes in ROS, regulates HIF-2α during hypoxia. [ABSTRACT FROM AUTHOR]

Ischemia-reperfusion (l/R) injury is a commonly encountered clinical problem in liver surgery and transplantation. The pathogenesis of I/R injury is multifactorial, but mitochondrial Ca2+ overload plays a central role. We have previously defined a novel pathway for mitochondrial Ca2+ handling and now further characterize this pathway and investigate a novel Ca2+-channel inhibitor, 2-aminoethoxydiphenyl borate (2-APB), for preventing hepatic I/R injury. The effect of 2-APB on cellular and mitochondrial Ca2+ uptake was evaluated in vitro by using 45Ca2+. Subsequently, 2-APB (2 mg/kg) or vehicle was injected into the portal vein of anesthetized rats either before or following 1 h of inflow occlusion to 70% of the liver. After 3 h of reperfusion, liver injury was assessed enzymatically and histologically. Hep O2 cells transfected with green fluorescent protein-tagged cytochrome c were used to evaluate mitochondrial permeability. 2-APB dose-dependently blocked Ca2+ uptake in isolated liver mitochondria and reduced cellular Ca2+ accumulation in Hep G2 cells. In vivo I/R increased liver enzymes 10-fold, and 2-APB prevented this when administered pre- or postischemia. 2-APB significantly reduced cellular damage determined by hematoxylin and eosin and terminal deoxynucleotidyl transferase dUTP-mediated nick-end labeling staining of liver tissue. In vitro I/R caused a dissociation between cytochrome c and mitochondria in Hep G2 cells that was prevented by administration of 2-APB. These data further establish the role of cellular Ca2+ uptake and subsequent mitochondrial Ca2+ overload in I/R injury and identify 2-APB as a novel pharmacological inhibitor of liver I/R injury even when administered following a prolonged ischemic insult. [ABSTRACT FROM AUTHOR]

Mcl-1 is an antiapoptotic member of the BcI-2 family that plays an important role in cell survival. We demonstrate that proteasome-dependent regulation of Mel-1 plays a critical role in renal tubular epithelial cell injury from cisplatin. Protein levels of Mcl-1 rapidly declined in a time-dependent manner following cisplatin treatment of LLC-PK1 cells. However, mRNA levels of Mcl-1 were not altered following cisplatin treatment. Expression of other antiapoptotic members of the Bcl-2 family such as Bcl-2 and BclxL was not affected by cisplatin treatment. Cisplatin-induced loss of Mcl-1 occurs at the same time as the mitochondrial release of cytochrome c, activation of caspase-3, and initiation of apoptosis. Treatment of cells with cycloheximide, a protein synthesis inhibitor, revealed rapid turnover of Mcl-1. In addition, treatment with cycloheximide in the presence or absence of cisplatin demonstrated that cisplatin-induced loss of Mcl-1 results from posttranslational degradation rather than transcriptional inhibition. Overexpression of Mel-1 protected cells from cisplatin-induced caspase-3 activation and apoptosis. Preincubating cells with the proteasome inhibitor MG-132 or lactacystin not only restored cisplatin-induced loss of Mcl-1 but also resulted in an accumulation of Mcl-1 that exceeded basal levels; however, Bcl-2 and BclxL levels did not change in response to MG-132 or lactacystin. The proteasome inhibitors effectively blocked cisplatin-induced mitochondriai release of cytochrome c, caspase-3 activation, and apoptosis. These studies suggest that proteasome regulation of Mcl-1 is crucial in the cisplatin-induced apoptosis via the mitochondrial apoptotic pathway and that Mcl-1 is an important therapeutic target in cisplatin injury to renal tubular epithelial cells. [ABSTRACT FROM AUTHOR]

We previously demonstrated that arachidonic acid (AA) inhibits epithelial Na channels (ENaC) through the cytochrome P-450 (CYP) epoxygenase-dependent pathway (34). In the present study, we tested the hypothesis that low Na intake suppresses the expression of CYP2C23, which is mainly responsible for converting AA to epoxyeicosatrienoic acid (EET) in the kidney (11) and attenuates the AA-induced inhibition of ENaC. Immunostaining showed that CYP2C23 is expressed in the Tamm-Horsfall protein (THP)-positive and aquaporin 2 (AQP2)- positive tubules. This suggests that CYP2C23 is expressed in the thick ascending limb (TAL) and collecting duct (CD). Na restriction significantly suppressed the expression of CYP2C23 in the TAL and CD. Western blot also demonstrated that the expression of CYP2C23 in renal cortex and outer medulla diminished in rats on Na-deficient diet (Na-D) but increased in those on high-Na diet (4%). Moreover, the content of 11, 12-epoxyeicosatrienoic acid (EET) decreased in the isolated cortical CD from rats on Na-D compared with those on a normal-Na diet (0.5%). Patch-clamp study showed that application of 15 µM AA inhibited the activity of ENaC by 77% in the CCD of rats on a Na-D for 3 days. However, the inhibitory effect of AA on ENaC was significantly attenuated in rats on Na-D for 14 days. Furthermore, inhibition of CYP epoxygenase with MS-PPOH increased the ENaC activity in the CCD of rats on a control Na diet. We also used microperfusion technique to examine the effect of MS-PPOH on Na transport in the distal nephron. Application of MS-PPOH significantly increased Na absorption in the distal nephron of control rats but had no significant effect on Na absorption in rats on Na-D for 14 days. We conclude that low Na intake downregulates the activity and expression of CYP2C23 and attenuates the inhibitory effect of AA on Na transport. [ABSTRACT FROM AUTHOR]

The cytochrome P-450 metabolite 20-HETE induces calcium-, endothelial-, and nitric oxide (NO)-dependent relaxation of bovine pulmonary arteries (PA). VEGF is an NO-dependent dilator of systemic arteries and plays a key role in maintaining the integrity of the pulmonary vasculature. We tested the effect of VEGF on PA diameter and tone and the contribution of cytochrome P-450 family 4 (CYP4) to vasoactive effects of VEGF. Bovine PA rings (1 mm in diameter) relaxed with VEGF (0.1-10 mM) in an endothelial- and eNOS-dependent manner. This response was blunted by pretreatment with the CYP4 inhibitor dibromododecynyl methyl sulfonamide (DDMS) as well as a mechanistically different CYP4 inhibitor N-hydroxy-N′-(4-butyl-2-methylphenyl)formamidine. PAs also increased in diameter by 6-12% in the presence of VEGF (10 nM), and this increase was attenuated by DDMS. In contrast to that shown in PAs, 20-HETE constricted bovine renal arteries and did not increase intracellular Ca2+ in renal artery endothelial cells as observed in bovine pulmonary artery endothelial cells (BPAECs). VEGF-evoked increases in intracellular Ca2+ concentration ([Ca2+]i) in BPAECs were blunted by treatment with DDMS. Both VEGF (10 nM) and 20-HETE (1-5 μM) stimulated NO release from cultured BPAECs, and once again VEGF-induced increases were attenuated by pretreating the cells with DDMS. We conclude that CYP4/20-HETE contributes to VEGF-stimulated NO release and vasodilation in bovine PAs. Given the unique expression of 20-HETE-forming CYP4 in BPAECs vs. systemic arterial endothelial cells, CYP4 may be an important mediator of endothelial-dependent vasoreactivity in PAs. [ABSTRACT FROM AUTHOR]

Cytochrome P-450 (CYP) epoxygenases and their arachidonic acid (AA) metabolites, the epoxyeicosatrienoic acids (EETs), have been shown to produce increases in postischemic function via ATP-sensitive potassium channels (KATP); however, the direct effects of EETs on infarct size (IS) have not been investigated. We demonstrate that two major regioisomers of CYP epoxygenases, 11,12-EET and 14,15-EET, significantly reduced IS in dogs compared to control (22.1 ± 1.8%), whether administered 15 min before 60 min of coronary occlusion (6.4 ± 1.9%, 11,12-EET; and 8.4 ± 2.4%, 14.15-EET) or 5 min before 3 h of reperfusion (8.8 ± 2.1%, 11,12-EET; and 9.7 ± 1.4%, 14,15-EET). Pretreatment with the epoxide hydrolase metabolite of 14,15-EET, 14,15-dihydroxyeicosatrienoic acid, had no effect. The protective effect of 11,12-EET was abolished (24.3 ± 4.6%) by the KATP channel antagonist glibenclamide. Furthermore, one 5-min period of ischemic preconditioning (IPC) reduced IS to a similar extent (8.7 ± 2.8%) to that observed with the EETs. The selective CYP epoxygenase inhibitor, N-methylsulfonyl-6-(2-propargyloxyphenyl)hexanamide (MS-PPOH), did not block the effect of IPC. However, administration of MS-PPOH concomitantly with N-methylsulfonyl-12, 12-dibromododec-11-enanide (DDMS), a selective inhibitor of endogenous CYP ω-hydroxylases, abolished the reduction in myocardial IS expressed as a percentage of area at risk (IS/AAR) produced by DDMS (4.6 ± 1.2%, DDMS; and 22.2 ± 3.4%, MS-PPOH + DDMS). These data suggest that 11,12-EET and 14,15-EET produce reductions in IS/AAR primarily at reperfusion. Conversely, inhibition of CYP epoxygenases and endogenous EET formation by MS-PPOH, in the presence of the CYP ω-hydroxylase inhibitor DDMS blocked cardioprotection, which suggests that endogenous EETs are important for the beneficial effects observed when CYP ω-hydroxylases are inhibited. Finally, the protective effects of EETs are mediated by cardiac KATP channels. [ABSTRACT FROM AUTHOR]

Epoxyeicosatrienoic acids (EETs) are cytochrome P-450 (CYP) metabolites synthesized from the essential fatty acid arachidonic acid to generate four regioisomers, 14,15-, 11,12-, 8,9-, and 5,6-EET. Cultured human coronary artery endothelial cells (HCAECs) contain endogenous EETs that are increased by stimulation with physiological agonists such as bradykinin. Because EETs are known to modulate a number of vascular functions, including angiogenesis, we tested each of the four regioisomers to characterize their effects on survival and apoptosis of HCAECs and cultured human lung microvascular endothelial cells (HLMVECs). A single application of physiologically relevant concentration of 14,15-, 11,12-, and 8,9-EET but not 5,6-EET (0.75–300 nM) promoted concentration-dependent increase in cell survival of HLMVECs and HCAECs after removal of serum. The lipids also protected the same cells from death via the intrinsic, as well as extrinsic, pathways of apoptosis. EETs did not increase intracellular calcium concentration ([Ca2+]1) or phosphorylate mitogen-activated protein kinase p44/42 when applied to these cells, and their protective action was attenuated by the phosphotidylinositol-3 kinase inhibitor wortmannin (10 μM) but not the cyclooxygenase inhibitor indomethacin (20 μM). Our results demonstrate for the first time the capacity of EETs to enhance human endothelial cell survival by inhibiting both the intrinsic, as well as extrinsic, pathways of apoptosis, an important underlying mechanism that may promote angiogenesis and endothelial survival during atherosclerosis and related cardiovascular ailments. [ABSTRACT FROM AUTHOR]

Reactive oxygen species (ROS) can cause cell injury and death via mitochondrial-dependent pathways, and supplementation with anti-oxidants has been shown to ameliorate these processes. The c-Jun NH2-terminal kinase (JNK) pathway has been shown to play a critical role in ROS-induced cell death. To determine if targeting catalase (CAT) to the mitochondria provides better protection than cytosolic expression against H2O2-induced injury, the following two approaches were taken: 1) adenoviral-mediated transduction was performed using cytosolic (CCAT) or mitochondrial (MCAT) CAT cDNAs and 2) stable cell lines were generated overexpressing CAT in mitochondria (n = 3). Cells were exposed to 250 μM H2O2, and cell survival, mitochondrial function, cytochrome c release, and JNK activity were analyzed. Although all viral transduced cells had a transient twofold increase in CAT activity, MCAT cells had significantly higher survival rates, the best mitochondrial function, and lowest JNK activity compared with CCAT and LacZ controls. The improved protection with MCAT was observed in primary type II lung epithelial cells and in transformed lung epithelial cells. In the three stable cell lines, cell survival directly correlated with extent of mitochondrial localization (r = 0.60572, P < 0.05) and not overall CAT activity (r = -0.4550 1, P < 0.05). Data indicate that targeting of antioxidants directly to the mitochondria is more effective in protecting lung epithelial cells against ROS-induced injury. This has important implications in antioxidant supplementation trials to prevent ROS-induced lung injury in critically ill patients. [ABSTRACT FROM AUTHOR]

Whether a cytochrome P-450 (CYP)-related endothelium-derived hyperpolarizing factor (EDHF), acting through calcium-activated potassium (KCa) channels, interacts with nitric oxide (NO) to regulate the basal diameter of human peripheral conduit arteries is unexplored in vivo. Radial artery diameter (echo tracking) and blood flow (Doppler) were measured, after oral aspirin (500 mg), in eight healthy volunteers during local infusion for 8 min of tetraethylammonium chloride (TEA; 9 μmol/min), as KCa channel inhibitor, and fluconazole (0.4 μmol/min), as CYP inhibitor, alone and in combination with NG-monomethyl-L-arginine (ʟ-NMMA; 8 μmol/min), as endothelial NO synthase inhibitor. Endothelium-independent dilatation was assessed by using sodium nitroprusside (SNP). Radial diameter was unaffected by ʟ-NMMA (0.4 ± 0.9%) and fluconazole (-1.6 ± 0.8%) but was decreased by TEA (-5.0 ± 1.0%), ʟ-NMMA + fluconazole (-5.3 ± 0.5%), and ʟ-NMMA + TEA (-9.9 ± 1.3%). These effects are still significant even when the concomitant decreases in blood flow induced by ʟ-NMMA (-24 ± 4%), TEA (-21 ± 3%), ʟ-NMMA + fluconazole (-26 ± 5%), and ʟ-NMMA + TEA (-35 ± 4%) were taken as covariate into analysis. Conversely, fluconazole alone slightly but not significantly increased radial flow (13 ± 6%). ʟ-NMMA alone or with TEA and with fluconazole enhanced radial artery dilatation to SNP, whereas TEA and fluconazole alone did not modify this response. These results confirm in humans the involvement of NO and KCa channels in the regulation of basal conduit artery diameter. Moreover, the synergistic effect of combined inhibition of NO synthesis and CYP on the decrease in radial diameter in the absence of such effect after ʟ-NMMA and fluconazole alone unmasks the role of CYP in this regulation and shows the presence of an interaction between NO and a CYP-related EDHF to maintain peripheral conduit artery diameter in vivo. Furthermore, the higher vasoconstrictor effect of TEA compared with fluconazole suggests that different KCa channel-dependent hyperpolarizing mechanisms could exist in conduit arteries. [ABSTRACT FROM AUTHOR]

Cytochrome P-450 (CYP) ω-hydroxylases and their arachidonic acid (AA) metabolite, 20-hydroxyeicosatetraenoic acid (20-HETE), produce a detrimental effect on ischemia-reperfusion injury in canine hearts, and the inhibition of CYP ω-hydroxylases markedly reduces myocardial infarct size expressed as a percentage of the area at risk (IS/AAR, %). In this study, we demonstrated that a specific CYP ω-hydroxylase inhibitor, N-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS), markedly reduced 20-HETE production during ischemia-reperfusion and reduced myocardial infarct size compared with control [19.5 ± 1.0% (control), 9.6 ± 1.5% (0.40 mg/kg DDMS), 4.0 ± 2.0% (0.81 mg/kg DDMS), P < 0.01]. In addition, 20-hydroxyeicosa-6(Z),15(Z)-dienoic acid (20-HEDE, a putative 20-HETE antagonist) significantly reduced myocardial infarct size from control [10.3 ± 1.3% (0.032 mg/kg 20-HEDE) and 5.9 ± 1.9% (0.064 mg/kg 20-HEDE), P < 0.05]. We further demonstrated that one 5-mm period of ischemic preconditioning (IPC) reduced infarct size to a similar extent as that observed with the high doses of DDMS and 20-HEDE, and the higher dose of DDMS given simultaneously with IPC augmented the infarct size reduction [9.9 ± 2.8% (IPC) to 2.5 ± 1.4% (0.81 mg/kg DDMS), P < 0.05] to a greater degree than that observed with either treatment alone. These results suggest an important negative role for endogenous CYP ω-hydroxylases and their product, 20-HETE, to exacerbate myocardial injury in canine myocardium. Furthermore, for the first time, this study demonstrates that the effect of IPC and the inhibition of CYP w-hydroxylase synthesis (DDMS) or its actions (20-HEDE) may have additive effects in protecting the canine heart from ischemia-reperfusion injury. [ABSTRACT FROM AUTHOR]

Apoptosis can be evoked by reactive oxygen species (ROS)-induced mitochondrial release of the proapoptotic factors cytochrome c and apoptosis-inducing factor (AIF). Because skeletal muscle is composed of two mitochondrial subfractions that reside in distinct subcellular regions, we investigated the apoptotic susceptibility of subsarcolemmal (SS) and intermyofibrillar (IMF) mitochondria. SS and IMF mitochondria exhibited a dose-dependent release of protein in response to H2O2 (0, 25, 50, and 100 µM). However, IMF mitochondria were more sensitive to H2O2 and released a 2.5-fold and 10-fold greater amount of cytochrome c and AIF, respectively, compared with SS mitochondria. This finding coincided with a 44% (P < 0.05) greater rate of opening (maximum rate of absorbance decrease, Vmax) of the protein release channel, the mitochondrial permeability transition pore (mtPTP), in IMF mitochondria. IMF mitochondria also exhibited a 47% (P < 0.05) and 60% (0.05 < P < 0.1) greater expression of the key mtPTP component voltage-dependent anion channel and cyclophilin D, respectively, along with a threefold greater cytochrome c content, but similar levels of AIF compared with SS mitochondria. Despite a lower susceptibility to H2O2-induced release, SS mitochondria possessed a 10-fold greater Bax-to-Bcl-2 ratio (P < 0.05), a 2.7-fold greater rate of ROS production, and an approximately twofold greater membrane potential compared with IMF mitochondria. The expression of the antioxidant enzyme Mn2+-superoxide dismutase was similar between subfractions. Thus the divergent protein composition and function of the mtPTP between SS and IMF mitochondria contributes to a differential release of cytochrome c and AIF in response to ROS. Given the relatively high proportion of IMF mitochondria within a muscle fiber, this subfraction is likely most important in inducing apoptosis when presented with apoptotic stimuli, ultimately leading to myonuclear decay and muscle fiber atrophy. [ABSTRACT FROM AUTHOR]

Elevated LPS and elevated cytochrome P-450 2E 1 (CYP2E1) in liver are two major independent risk factors in alcoholic liver disease. We investigated possible synergistic effects of the two risk factors in causing oxidative stress and liver injury. Sprague-Dawley rats were injected intraperitoneally with pyrazole (inducer of CYP2E1) for 2 days, and then LPS was injected via tail vein. Other rats were treated with pyrazole alone or LPS alone or saline. Eight hours later, blood was collected and livers were excised. Pathological evaluation showed severe inflammatory responses and necroses only in liver sections from rats in the pyrazole plus LPS group; blood transaminase levels were significantly elevated only in the combination group. Activities of caspase-3 and -9 and positive terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling staining were highest in the LPS alone and the LPS plus pyrazole group, with no significant difference between the two groups. Lipid peroxidation and protein carbonyls in liver homogenate as well as in situ superoxide production were maximally elevated in the LPS plus pyrazole group. Levels of nitrite plus nitrate and inducible nitric oxide (NO) synthase (iNOS) content were comparably elevated in LPS alone and the LPS plus pyrazole group; however, 3-nitrotyrosine adducts were elevated in the combined group but not the LPS group. It is likely that LPS induction of iNOS, which produces NO, coupled to pyrazole induction of CYP2E1 which produces superoxide, sets up conditions for maximal peroxynitrite formation and production of 3-nitrotyrosine adducts. CYP2E1 activity and content were elevated in the pyrazole and the LPS plus pyrazole groups. Immunohistochemical staining indicated that distribution of CYP2E1 was in agreement with that of necrosis and production of superoxide. These results show that pyrazole treatment enhanced LPS-induced necrosis, not apoptosis. The enhanced liver necrosis appears to involve an increase in oxidative and nitrosative stress generated by the combination of LPS plus elevated CYP2E1 levels. [ABSTRACT FROM AUTHOR]

Studies were performed to further characterize the male-specific hepatic recombinant microsomal vitamin D 25-fiydroxlase CYP2C11, expressed in baculovirus-infected insect cells, and determine whether it is also a vitamin D 24-hydroxylase. 25- and 24-hydroxylase activities were compared with those of 10 other recombinant hepatic microsomal cytochrome P-450 enzymes expressed in baculovirus-infected insect cells. Each of them 25-hydroxylated vitamin D2, vitamin D3, 1α-hydroxyvitamin D2 (1αOHD2), and 1α-hydroxyvitamin D3 (1αOHD3). CYP2C11 had the greatest activity with these substrates, except vitamin D3, which had the same activity as four of the other enzymes. The descending order of 25-hydroxylation by CYP2C11 was 1αOHD3 > 1αOHD2 > vitamin D2 > vitamin D3. Each of the recombinant cytochrome P-450 enzymes 24-hydroxylated 1αOHD2. CYP2C11 had the greatest activity. 24-Hydroxylation of 1αOHD3 was very low, and there was none with vitamin D3. Only CYP2C11 24-hydroxylated vitamin D2. Structures of vitamin D metabolites, including 24-hydroxyvitamin D2, 1,24(S)-dihydroxyvitamin D2, and 1,24-dihydroxyvitamin D3, were confirmed by HPLC and gas chromatography retention times and characteristic mass spectrometric fragmentation patterns. In male rats, hypophysectomy significantly reduced body weight, liver weight, hepatic CYP2C11 mRNA expression, and 24- and 25-hydroxylation of 1αOHD2. Expression of CYP2J3 and CYP2R1 mRNA did not change. In male rat hepatocytes, CYP2C11 mRNA expression and 24- and 25-hydroxylation were significantly reduced after culture for 24 h compared with uncultured cells. Expression of CYP2J3 and CYP2R1 either increased or did not change. It is concluded that CYP2C11 is a male-specific hepatic microsomal vitamin D 25-hydroxylase that hydroxylates vitamin D2, vitamin D3, 1αOHD2, and 1αOHD3. CYP2C11 is also a vitamin D 24-hydroxylase. [ABSTRACT FROM AUTHOR]

Cd2+ induces apoptosis of kidney proximal tubule (PT) cells. Mitochondria play a pivotal role in toxic compound induced apoptosis by releasing cytochrome c. Our objective was to investigate the mechanisms underlying Cd2+-induced cytochrome c release from mitochondria in rat PT cells. Using Hoechst 33342 or MTT assay, 10 µM Cd2+ induced ∼5-10% apoptosis in PT cells at 6 and 24 h, which was associated with cytochrome c and apoptosisinducing factor release at 24 h only. This correlated with previously described maximal intracellular Cd2+ concentrations at 24 h, suggesting that elevated Cd 2+ may directly induce mitochondrial liberation of proapoptotic factors. Indeed, Cd2+ caused swelling of energized isolated kidney cortex mitochondria (EC50 ∼9 µM) and cytochrome c release, which were independent: of permeability transition pore (PTP) opening since PTP inhibitors cyclosporin A or bongkrekic acid had no effect. On the contrary, Cd2+ inhibited swelling and cytochrome c release induced by PTP openers (PO43- or H2O2 + Ca2+). The mitochondrial Ca2+ uniporter (MCU) played a key role in mitochondrial damage: 1) MCU inhibitors (La3+, ruthenium red, Ru360) prevented swelling and cytochrome c release; and 2) ruthenium red attenuated Cd2+ inhibition of PO43- -induced swelling. Using the Cd2+-sensitive fluorescent indicator FluoZin-1, Cd2+ was also taken up by mitoplasts. The aquaporin inhibitor AgNO3 abolished Cd2+-induced swelling of mitoplasts. This could be partially mediated by activation of the mitoplast-enriched water channel aquaporin-8. Thus cytosolic Cd2+ concentrations exceeding a certain threshold may directly cause mitochondrial damage and apoptotic development by interacting with MCU and water channels in the inner mitochondrial membrane. [ABSTRACT FROM AUTHOR]

Endothelium-derived nitric oxide (NO) plays an important role in the regulation of vascular tone. Lack of NO bioavailability can result in cardiovascular disease. NO bioavailability is determined by its rates of generation and catabolism; however, it is not known how the NO catabolism rate is regulated in the vascular wall under normoxic, hypoxic, and anaerobic conditions. To investigate NO catabolism under different oxygen concentrations, studies of NO and O2 consumption by the isolated rat aorta were performed using electrochemical sensors. Under normoxic conditions, the rate of NO consumption in solution was enhanced in the presence of the rat aorta. Under hypoxic conditions, NO consumption decreased in parallel with the O2 concentration. Like the inhibition of mitochondrial respiration by NO, the inhibitory effects of NO on aortic O2 consumption increased as O2 concentration decreased. Under anaerobic conditions, however, a paradoxical reacceleration of NO consumption occurred. This increased anaerobic NO consumption was inhibited by the cytochrome c oxidase inhibitor NaCN but not by the free iron chelator deferoxamine, the flavoprotein inhibitor diphenylene iodonium (10 µM), or superoxide dismutase (200 U/ml). The effect of O2 on the NO consumption could be reproduced by purified cytochrome c oxidase (CcO), implying that CcO is involved in aortic NO catabolism. This reduced NO catabolism at low O2 tensions supports the maintenance of effective NO levels in the vascular wall, reducing the resistance of blood vessels. The increased anaerobic NO catabolism may be important for removing excess NO accumulation in ischemic tissues. [ABSTRACT FROM AUTHOR]

Mutations in the gene SURF1 prevent synthesis of cytochrome-c oxidase (COX)-specific assembly protein and result in a fatal neurological disorder, Leigh syndrome. Because this severe COX deficiency presents with barely detectable changes of cellular respiratory rates under normoxic conditions, we analyzed the respiratory response to low oxygen in cultured fibroblasts harboring SURF1 mutations with high-resolution respirometry. The oxygen kinetics was quantified by the partial pressure of oxygen (PO2) at half-maximal respiration rate (P50) in intact coupled cells and in digitonin-permeabilized uncoupled cells. In both cases, the P50 in patients was elevated 2.1- and 3.3-fold, respectively, indicating decreased affinity of COX for oxygen. These results suggest that at physiologically low intracellular PO2, the depressed oxygen affinity may lead in vivo to limitations of respiration, resulting in impaired energy provision in Leigh syndrome patients. [ABSTRACT FROM AUTHOR]

Tetracyclines exhibit significant anti-inflammatory properties in a variety of rheumatologic and dermatologic conditions. They have also been shown to inhibit apoptosis in certain neurodegenerative disorders. Because ischemic renal injury is characterized by both apoptosis and inflammation, we investigated the therapeutic potential of tetracyclines in a rat model of renal ischemiareperfusion. Male Sprague-Dawley rats underwent bilateral renal artery clamp for 30 min followed by reperfusion and received either minocycline or saline for 36 h before ischemia. Minocycline reduced tubular cell apoptosis 24 h after ischemia as determined by terminal transferase-mediated dUTP nick end-labeling staining and nuclear morphology. It also decreased cytochrome c release into the cytoplasm and reduced upregulation of p53 and Bax after ischemia. The minocycline-treated group showed a significant reduction in tubular injury and cast formation. In addition, minocycline reduced the number of infiltrating leukocytes, decreased leukocyte chemotaxis both in vitro and ex vivo, and downregulated the expression of ICAM-1. Serum creatinine 24-h postischemia was significantly reduced in the minocycline-treated group. We conclude that minocycline has potent antiapoptotic and anti-inflammatory properties and protects renal function in this model of ischemia-reperfusion. Tetracyclines are among the safest and best-studied antibiotics. They are thus attractive candidates for the therapy of human ischemic acute renal failure. [ABSTRACT FROM AUTHOR]

Cardiomyocyte apoptosis contributes to cell death during myocardial infarction. One of the factors that regulate the degree of apoptosis during ischemia is the amino acid taurine. To study the mechanism underlying the beneficial effect of taurine, we examined the interaction between taurine and mitochondria-mediated apoptosis using a simulated ischemia model with cultured rat neonatal cardiomyocytes sealed in closed flasks. Exposure to medium containing 20 mM taurine reduced the degree of apoptosis following periods of ischemia varying from 24 to 72 h. In the untreated group, simulated ischemia for 24 h led to mitochondrial depolarization accompanied by cytochrome c release. The apoptotic cascade was also activated, as evidenced by the activation of caspase-9 and -3. Taurine treatment had no effect on mitochondrial membrane potential and cytochrome c release; however, it inhibited ischemia-induced cleavage of caspase-9 and -3. Taurine loading also suppressed the formation of the Apaf-1/ caspase-9 apoptosome and the interaction of caspase-9 with Apaf-1. These findings demonstrate that taurine effectively prevents myocardial ischemia-induced apoptosis by inhibiting the assembly of the Apaf-1/caspase-9 apoptosome. [ABSTRACT FROM AUTHOR]

Previously, we demonstrated that both 150 mM KCl and alkaline pH inhibit cytochrome c-mediated activation of procaspase-3 in a unique manner. To determine the mechanism of inhibition, we analyzed the effect of KCl and alkaline pH on the formation of apoptosomes (a large complex consisting of cytochrome c, Apaf-1, and procaspase-9/caspase-9) in vitro. Our results suggest that an initial ∼700-kDa apoptosome matures through a 1.4-MDa intermediate before a ∼700-kDa apoptosome is reformed and procaspase-3 is activated. We further demonstrate that 150 mM KCl interferes with the conversion of the initial ∼700-kDa apoptosome to the 1.4-MDa intermediate, while alkaline pH "traps" the apoptosome in the 1.4-MDa intermediate. Analysis of the cleaved state of procaspase-9 and procaspase-3 suggests that the 1.4-MDa intermediate may be required for cleavage of procaspase-9. Consistent with these results, in vivo data suggest that blocking acidification during the induction of apoptosis inhibits activation of procaspase-3. On the basis of these results, we propose a model of apoptosome maturation. [ABSTRACT FROM AUTHOR]

Rajapakse, Niwanthi W., Richard J. Roman, John R. Falck, Jeremy J. Oliver, and Roger G. Evans. Modulation of V1-receptor- mediated renal vasoconstriction by epoxyeicosatrienoic acids. Am J Physiol Regul Integr Comp Physiol 287: R181-R187, 2004. First published February 26, 2004; 10.1152/ajpregu.00555.2002.---This study examined the effects of renal arterial infusion of a selective cytochrome P-450 epoxygenase inhibitor, N-methylsulfonyl-6- (2-propargyloxyphenyl)hexanamide (MS-PPOH; 2 mg/kg plus 1.5 mg·kg-l·h-1), on renal hemodynamic responses to infusions of [Phe2,Ile3,Orn8]vasopressin and ANG II into the renal artery of anesthetized rabbits. MS-PPOH did not affect basal renal blood flow (RBF) or cortical or medullary blood flow measured by laser-Doppler flowmetry (CLDF/MLDF). In vehicle-treated rabbits, [Phe2,Ile3,Orn8]- vasopressin (30 ng·kg-1·min-1) reduced MLDF by 62 ± 7% but CLDF and RBF were unaltered. In MS-PPOH-treated rabbits, RBF and CLDF fell by 51 ± 8 and 59 ± 13%, respectively, when [Phe2,Ile3,Orn8]vasopressin was infused. MS-PPOH had no significant effects on the MLDF response to [Phe2,Ile3,Orn8]vasopressin (43 ± 9% reduction). ANG II (20 ng·kg-1·min-1) reduced RBF by 45 ± 10% and CLDF by 41 ± 14%, but MLDF was not significantly altered. MS-PPOH did not affect blood flow responses to ANG II. Formation of epoxyeicosatrienoic acids (EETs) and dihydroxyeicosa- trienoic acids (DiHETEs) was 49% lower in homogenates prepared from the renal cortex of MS-PPOH-treated rabbits than from vehicle- treated rabbits. MS-PPOH had no effect on the renal formation of 20-hydroxyeicosatetraenoic acid (20-HETE). Incubation of renal cortical homogenates from untreated rabbits with [Phe2,Ile3,Orn8]vasopressin (0.2–20 ng/ml) did not affect formation of EETs, DiHETEs, or 20-HETE. These results do not support a role for de novo EET synthesis in modulating renal hemodynamic responses to ANG II. However, EETs appear to selectively oppose V1-receptor-mediated vasoconstriction in the renal cortex but not in the medullary circulation and contribute to the relative insensitivity of medullary blood flow to V1-receptor activation. angiotensin II; cytochrome P-450 enzyme system; kidney cortex; kidney medulla; renal circulation; vasopressin [ABSTRACT FROM AUTHOR]

Dos Santos, Elisabete A., Annette J. Dahly-Vernon, Kimberly M. Hoagland, and Richard J. Roman. Inhibition of the formation of EETs and 20-HETE with 1-aminobenzotriazole attenuates pressure natriuresis. Am J Physiol Regul Integr Comp Physiol 287: R58-R68, 2004. First published March 18, 2004; 10.1152/ajpregu. 00713.2003.—This study examined the effects of chronic blockade of the renal formation of epoxyeicosatrienoic acids and 20-hydroxyei- cosatetraenoic acid with 1-aminobenzotriazole (ABT; 50 mg·kg-1· day-1 ip for 5 days) on pressure natriuresis and the inhibitory effects of elevations in renal perfusion pressure (RPP) on Na+-K+-ATPase activity and the distribution of the sodium/hydrogen exchanger (NHE)-3 in the proximal tubule of rats. In control rats (n = 15), sodium excretion rose from 2.3 ± 0.4 to 19.4 ± 1.8 μeq·min-1·g kidney weight-1 when RPP was increased from 114 ± 1 to 156 ± 2 mmHg. Fractional excretion of lithium rose from 28 ± 3 to 43 ± 3% of the filtered load. Chronic treatment of the rats with ABT for 5 days (n = 8) blunted the natriuretic response to elevations in RPP by 75% and attenuated the increase in fractional excretion of lithium by 45%. In vehicle-treated rats, renal Na+-K+-ATPase activity fell from 31 ± 5 to 19 ± 2 μmol Pi·mg protein-1·h-1 and NHE-3 protein was internalized from the brush border of the proximal tubule after an elevation in RPP. In contrast, Na+-K+-ATPase activity and the distribution of NHE-3 protein remained unaltered in rats treated with ABT. These results suggest that cytochrome P-450 metabolites of arachidonic acid contribute to pressure natriuresis by inhibiting Na+-K+-ATPase activity and promoting internalization of NHE-3 protein from the brush border of the proximal tubule. 20-hydroxyeicosatetraenoic acid; epoxyeicosatrienoic acids; sodium/ hydrogen exchanger-3; sodium-potassium-adenosinetriphosphatase; kidney; proximal tubule; renal hemodynamics [ABSTRACT FROM AUTHOR]

Ischemia and reperfusion result in mitochondrial dysfunction, with decreases in oxidative capacity, loss of cytochrome c, and generation of reactive oxygen species. During ischemia of the isolated perfused rabbit heart, subsarcolemmal mitochondria, located beneath the plasma membrane, sustain a loss of the phospholipid cardiolipin, with decreases in oxidative metabolism through cytochrome oxidase and the loss of cytochrome c. We asked whether additional injury to the distal electron chain involving cardiolipin with loss of cytochrome c and cytochrome oxidase occurs during reperfusion. Reperfusion did not lead to additional damage in the distal electron transport chain. Oxidation through cytochrome oxidase and the content of cytochrome c did not further decrease during reperfusion. Thus injury to cardiolipin, cytochrome c, and cytochrome oxidase occurs during ischemia rather than during reperfusion. The ischemic injury leads to persistent defects in oxidative function during the early reperfusion period. The decrease in cardiolipin content accompanied by persistent decrements in the content of cytochrome c and oxidation through cytochrome oxidase is a potential mechanism of additional myocyte injury during reperfusion. [ABSTRACT FROM AUTHOR]

The objective of this work was to evaluate the effects of testosterone (T) and 17β-estradiol (E2) on coronary microvascular endothelial cells (CMECs) of male and female rats. To analyze the short-terra effects of such sex steroid hormones on intracellular Ca2+ concentration ([Ca2+]i) kinetics, we used the chelating agent fura-2 acetoxymethyl ester. We also explored the possibility of testosterone aromatization by using selective inhibitors of the aromatase enzyme cytochrome P-450 aromatase (P450arom ), aminoglutethimide (4 µM), and 4-hydroxyandrostenedione (4 µM). The presence of P450arom was investigated by immunocytochemical and immunoblot assays using peptide-generated polyclonal antibodies raised against a 20-amino acid synthetic fragment of rat P450arom and by in situ hybridization to locate the aromatase mRNA in such cells. The activity of P450arom was demonstrated by the stereospecific loss of the tritium atom of [l[β-³H]androstenedione. Our results indicate that both T and E2 induced a rapid increase in [Ca2+]i. The fact that the effects of E: and T were carried out within milliseconds suggests that they were exerted at the membrane level and not through intracellular receptors. The possibility of involvement of PLC-β in these effects is suggested because U-73122 (a PLC inhibitor) blocked the effects of both T and E2. Immunocytochemical assays indicated the expression of androgenic and estrogenic receptors in these cells. The effects of T were blocked by the selective aromatase inhibitors. We also demonstrated membrane association of P450arom, expression of the ovary-specific mRNA after in sim hybridization, and E2 formation resulting from a significant activity of P450arom in CMECs. There were no gender-based differences. [ABSTRACT FROM AUTHOR]

Recent studies indicate that cytochrome c is released early in apoptosis without loss of integrity of the mitochondrial outer membrane in some cell types. The high-conductance mitochondrial apoptosis-induced channel (MAC) forms in the outer membrane early in apoptosis of FL5.12 cells. Physiological (micromolar) levels of cytochrome c alter MAC activity, and these effects are referred to as types 1 and 2. Type 1 effects are consistent with a partitioning of cytochrome c into the pore of MAC and include a modest decrease in conductance that is dose and voltage dependent, reversible, and has an increase in noise. Type 2 effects may correspond to ’plugging’ of the pore or destabilization of the open state. Type 2 effects are a dose-dependent, voltage-independent, and irreversible decrease in conductance. MAC is a heterogeneous channel with variable conductance. Cytochrome c affects MAC in a pore size-dependent manner, with maximal effects of cytochrome c on MAC with conductance of 1.9–5.4 nS. The effects of cytochrome c, RNase A, and high salt on MAC indicate that size, rather than charge, is crucial. The effects of dextran molecules of various sizes indicate that the pore diameter of MAC is slightly larger than that of 17-kDa dextran, which should be sufficient to allow the passage of 12-kDa cytochrome c. These findings are consistent with the notion that MAC is the pore through which cytochrome c is released from mitochondria during apoptosis. [ABSTRACT FROM AUTHOR]

Cytochrome c expression and mitochondrial biogenesis can be invoked by elevated intracellular Ca2+ in muscle cells. To characterize the potential role of Ca2+ as a messenger involved in mitochondrial biogenesis in muscle, we determined the effects of the Ca2+ ionophore A-23187 on the expression of nuclear- and mitochondrially encoded genes. Treatment of myotubes with 1 μM A-23187 for 48–96 h increased nuclear-encoded β-subunit F1ATPase and malate dehydrogenase (MDH) mRNA levels by 50100% (P < 0.05) but decreased mRNA levels of glutamate dehydrogenase (GDH) by 19% (P < 0.05). mRNA levels of the cytochrome c oxidase (COX) nuclear-encoded subunits IV, Vb, and VIc were unchanged, whereas the mitochondrially encoded subunits COX II and COX III were decreased by 30 and 70%, respectively (P < 0.05). This was paralleled by a 20% decrease (P < 0.05) in COX activity. These data suggest that cytoplasmic Ca2+ differentially regulates the mRNA level of nuclear and mitochondrial genes. The decline in COX II and III mRNA may be mediated by Tfam, because A-23187 modestly reduced Tfam levels by 48 h. A-23187 induced timedependent increases in Egr-I mRNA, along with the activation of ERK1/2 and AMP-activated protein kinase. MEK inhibition with PD-98059 attenuated the increase in Egr-1 mRNA. A-23187 also increased Egr-l, serum response factor, and Spl protein expression, transcription factors implicated in mitochondrial biogenesis. Egr-1 overexpression increased nuclear-encoded cytochrome c transcriptional activation by 1.5-fold (P < 0.05) and reduced GDH mRNA by 37% (P < 0.05) but had no effect on MDH or β-subunit F1ATPase mRNA. These results indicate that changes in intracellular Ca2+ can modify mitochondrial phenotype, in part via the involvement of Egr- 1. [ABSTRACT FROM AUTHOR]

Investigates the hypothesis that the rat pre-Botzinger complex (PBC) exhibited reduced cytochrome odixase activity on post-natal (P) days and especially on P12. Effect of carotid body denervation on PBC; Loss in body weight in all animals; Reduction in PBC somal size when the surgery was between P2 and P7; Prolongation of the maturational process.

Examines the influence of polyamine depletion in preventing capotothecin-induced apoptosis. Inhibition of the release of cythocrome; Regulation of apoptosis in mammalian cells; Role of the mitochondria.

Analyzes the impact of cytochrome P-450 arachidonate metabolites on endothelin signaling in the proximal renal tubule of rats. Ion transport in freshly isolated proximal tubules; Measurement by gas chromatography and mass spectrometry; Proximal tubule production of 20-hydroxyeicosatetraenoic acid.

Focuses on a study which examined whether cytochrome enzymes CYP2E1 and CYP4A can initiate hepatocellular injury and secondary recruitment of inflammation. Regulation of the cytochromes; Overview of the cytochromes as a source of prooxidants in liver cells; Discussion on CYP-mediated upregulation of antioxidant enzymes; Conclusions.

Evaluates the possible role that vasoconstrictor metabolites of cytochrome P-450 w-hydroxylase plays in oxygen-induced constriction of arterioles in the rat skeletal muscle circulation. Response of third-order arterioles in the rat cremaster muscle to superfusion; Immunohistochemical localization of CYP4A proteins.

Studies the role of cytochrome P-450-derived eicosanoids in the lung. Metabolic pathways where free arachidonic acid is available for oxidation; Background of P-450 cytochromes; Biological activity of the eicosanoids in the lung; Tools to explore the functional significance of P-450 expression; Conclusions.

Presents a study which evaluated the contribution of a type of cytochrome in modulating the reactivity of cremaster muscle arterioles in rats on salt diet and with hypertension. Materials and methods; Results; Discussion of the results.

Studies the levels of P-450 hydroxyeicosatetraenoic acids (HETE) exiting the rabbit perfused kidney in response to hormonal stimulation. Biological properties of the stereoisomers of 16, 17, 17 and 19-HETEs in terms of vasoactivity and potential effects on ion transport; Inhibition of proximal tubule adenosine triphosphatase activity.