Rat Lung Mitochondria Research Articles

Inhibitory Effects of Scutellaria baicalensis Root Extract on Linoleic Acid Hydroperoxide-induced Lung Mitochondrial Lipid Peroxidation and Antioxidant Activities.

In this study, we evaluated the ability of Scutellaria baicalensis Georgi to protect lipid-peroxidation (LPO) in lung tissue after free radical-induced injury. We prepared S. baicalensis root (SBR) extracts using different solvents. The total flavonoid and total phenol contents of each extract were measured, and the ROS damage protection was evaluated by analyzing linoleic acid hydroperoxide (LHP)-induced LPO in rat lung mitochondria. Moreover, evaluating diphenylpicrylhydrazyl (DPPH), hydrogen peroxide, superoxide anion radical, and hydroxyl radical scavenging abilities and using metal chelating assays were used to determine in vitro antioxidant activity. The ethyl acetate (EtOAc) extract showed high ROS scavenging ability, and four compounds were subsequently isolated and purified from this extract: baicalin, baicalein, wogonin, and oroxylin A. Baicalein in rat lung mitochondria the most significant LHP-induced LPO inhibition was shown and extracted with EtOAc that contained the highest amount of baicalein. Thus, baicalein and the EtOAc extract of SBR may be efficient in conferring ROS damage protection and inhibiting LHP-induced LPO in rat lung mitochondria. Additional studies are warranted to investigate their use as antioxidant therapy for respiration infections, nutrition supplements, and lead compounds in pharmaceuticals.

Heme Oxygenase-1/Carbon Monoxide-regulated Mitochondrial Dynamic Equilibrium Contributes to the Attenuation of Endotoxin-induced Acute Lung Injury in Rats and in Lipopolysaccharide-activated Macrophages.

Sepsis-associated acute lung injury remains the major cause of mortality in critically ill patients and is characterized by marked oxidative stress and mitochondrial dysfunction. Mitochondrial dynamics are indispensable for functional integrity. Additionally, heme oxygenase (HO)-1/carbon monoxide conferred cytoprotection against end-organ damage during endotoxic shock. Herein, we tested the hypothesis that HO-1/carbon monoxide played a critical role in maintaining the dynamic process of mitochondrial fusion/fission to mitigate lung injury in Sprague-Dawley rats or RAW 264.7 macrophages exposed to endotoxin. The production of reactive oxygen species, the respiratory control ratio (RCR), and the expressions of HO-1 and mitochondrial dynamic markers were determined in macrophages. Concurrently, alterations in the pathology of lung tissue, lipid peroxidation, and the expressions of the crucial dynamic proteins were detected in rats. Endotoxin caused a 31% increase in reactive oxygen species and a 41% decrease in RCR levels (n = 5 per group). In parallel, the increased expression of HO-1 was observed in lipopolysaccharide-stimulated macrophages, concomitantly with excessive mitochondrial fission. Furthermore, carbon monoxide-releasing molecule-2 or hemin normalized mitochondrial dynamics, which were abrogated by zinc protoporphyrin IX. Additionally, impaired mitochondrial dynamic balance was shown in Sprague-Dawley rats that received lipopolysaccharide, accompanied by pathologic injury, elevated malondialdehyde contents, decreased manganese superoxide dismutase activities, and lowered RCR levels in rat lung mitochondria. However, the above parameters were augmented by zinc protoporphyrin IX and were in turn reversed by hemin. The HO-1/carbon monoxide system modulated the imbalance of the dynamic mitochondrial fusion/fission process evoked by lipopolysaccharide and efficiently ameliorated endotoxin-induced lung injury in vivo and in vitro.

L-NAME co-treatment prevent oxidative damage in the lung of adult Wistar rats treated with vitamin A supplementation

Based on the fact that vitamin A in clinical doses is a potent pro-oxidant agent to the lungs, we investigated here the role of nitric oxide (NO•) in the disturbances affecting the lung redox environment in vitamin A-treated rats (retinol palmitate, doses of 1000-9000 IU•kg(-1)•day(-1)) for 28 days. Lung mitochondrial function and redox parameters, such as lipid peroxidation, protein carbonylation and the level of 3-nytrotyrosine, were quantified. We observed, for the first time, that vitamin A supplementation increases the levels of 3-nytrotyrosine in rat lung mitochondria. To determine whether nitric oxide (NO •) or its derivatives such as peroxynitrite (ONOO-) was involved in this damage, animals were co-treated with the nitric oxide synthase inhibitor L-NAME (30 mg•kg(-1), four times a week), and we analysed if this treatment prevented (or minimized) the biochemical disturbances resulting from vitamin A supplementation. We observed that L-NAME inhibited some effects caused by vitamin A supplementation. Nonetheless, L-NAME was not able to reverse completely the negative effects triggered by vitamin A supplementation, indicating that other factors rather than only NO• or ONOO- exert a prominent role in mediating the redox effects in the lung of rats that received vitamin A supplementation.

Changes in cytochrome c oxidase and NO in rat lung mitochondria following iron overload

In this study, the effects of iron on cytochrome c oxidase (CcO) in rat lung mitochondria were examined. Similar to liver mitochondria, iron accumulated considerably in lung mitochondria (more than 2‐fold). Likewise, the reactive oxygen species and nitric oxide (NO) content of mitochondria were increased by more than 50% and 100%, respectively. NO might be produced by nitric oxide synthase (NOS), eNOS and iNOS type, with particular contribution by NOS in mitochondria. The respiratory control ratio of ironoverloaded lung mitochondria dropped to nearly 50% due to increased state 4. Likewise, cytochrome c oxidase activity was lowered significantly to approximately 50% due to excess iron. Real‐time PCR revealed that the expression of isoforms 1 and 2 of subunit IV of CcO was enhanced greatly under excess iron conditions. Taken together, these results show that oxidative phosphorylation within lung mitochondria may be influenced by iron overload through changes in cytochrome c oxidase and NO.

Constitutive and Inducible Cytochromes P450 in Rat Lung Mitochondria: Xenobiotic Induction, Relative Abundance, and Catalytic Properties

The presence of xenobiotic-inducible CYP1A1, 2B1/2, and 3A1/2 in rat lung mitochondria was investigated using mitochondrial preparations of defined purity. The mitochondrial P450 content in uninduced lung was 1.5-fold higher compared to microsomes. Administration of BNF induced the P450 contents by twofold in both mitochondrial and microsomal membrane fractions. BNF treatment induced EROD activity to about 40-fold in the microsomal fraction and 25-fold in the mitochondrial fraction. The microsomal induction was observed at 4 days of BNF treatment, while the mitochondrial induction required 10 days of treatment. Consistent with the activity profile, Western blot analysis showed the presence of CYP1A1 antibody reactive protein only in lung mitochondria from BNF-treated rats. BNF administration also caused a 50 to 80% reduction in the CYP2B1/2-associated PROD and BROD activities and CYP3A1/2-associated ERND activity in both mitochondria and microsomes. There was also a parallel reduction in the antibody reactive CYP2B1/2 and 3A1/2 proteins in both of these membrane fractions. Administration of DEX for 4 days induced mitochondrial and microsomal ERND activity by 1.7- and 2.5-fold, respectively. Mitochondrial EROD activity was inhibited by antibodies to P450MT2, as well as Adx, but not by antibody against P450 reductase, indicating the mitochondrial localization of CYP1A1. Protease protection and alkaline extraction experiments indicated that CYP1A1 associated with lung mitochondria is localized inside the inner membrane and exists as a membrane extrinsic protein. In summary, this is probably the first report of inducible P450s in rat lung mitochondria, and our results suggest a possible functional role for these mitochondrial enzymes in xenobiotic metabolism.

Synthesis of phosphatidylcholine and phosphatidylglycerol in rat lung mitochondria.

The mitochondrial fraction of adult rat lung contains choline phosphotransferase (EC 2.7.8.2) activity which can not be explained by microsomal contamination estimated on the basis of marker enzyme distribution. Mitochondrial (14C)glycerol-3-phosphate incorporation into PC (phosphatidylcholine) can be distinguished from the microsomal incorporation by different sensitivity to N-ethylmaleimide inhibition. The data indicate that rat lung mitochondria have the intrinsic capability to synthesize PC. Both synthesis of PC and PG (phosphatidylglycerol) are susceptible to isotonic tryptic attack against the cytoplasmic face of isolated rat lung mitochondria, suggesting the outer membrane location of crucial activities involved in the formation of these phospholipids. Rat liver mitochondria are different from rat lung mitochondria with respect to their capability to synthesize PC, their rate of (14C)glycerol-3-phosphate incorporation into PG as well as the submitochondrial site of PG formation.

Characterization of CDPdiacylglycerol hydrolase in mitochondrial and microsomal fractions from rat lung.

CDPdiacylglycerol pyrophosphatase (E.C. 3.6.1.26) activity has been examined in rat lung mitochondrial and microsomal fractions. While the mitochondrial hydrolase exhibited a broad pH optimum from pH 6-8, the microsomal activity decreased rapidly above pH 6.5. Apparent Km values of 36.2 and 23.6 microM and Vmax values of 311 and 197 pmol.min-1.mg protein-1 were observed for the mitochondrial and microsomal preparations, respectively. Addition of parachloromercuriphenylsulphonic acid led to a marked inhibition of the microsomal fraction but slightly stimulated the mitochondrial activity at low concentrations. Mercuric ions were inhibitory with both fractions. Although biosynthetic reactions utilizing CDPdiacylglycerol require divalent cations, addition of Mg2+, Mn2+, Ca2+, Zn2+, Co2+, and Cu2+ all inhibited the catabolic CDPdiacylglycerol hydrolase activity in both fractions. EDTA and EGTA also produced an inhibitory effect, especially with the mitochondrial fraction. Although addition of either adenine or cytidine nucleotides led to a decrease in activity with both fractions, the marked susceptibility to AMP previously reported for this enzyme in Escherichia coli membranes, guinea pig brain lysosomes, and pig liver mitochondria was not observed. These results indicate that rat lung mitochondria and microsomes contain specific CDPdiacylglycerol hydrolase activities, which could influence the rate of formation of phosphatidylinositol and phosphatidylglycerol for pulmonary surfactant.

Binding of basic drugs to rat lung mitochondria.

The role of the mitochondria in the accumulation of basic amine drugs in the rat lung was studied. Drug binding to the mitochondria was rapid and reached maximum levels after 2.5 min of incubation. Lipophilic basic drugs accumulated in the mitochondria more than nonlipophilic basic drugs and nonbasic drugs, and the accumulation was dose dependent. Schatchard plots revealed at least two independent sets of binding sites for basic drugs in the mitochondria. The binding was competitively inhibited by other basic drugs but not nonbasic drugs. The degree of inhibition by competing basic drugs was correlated with their lipid solubilities. These findings with isolated mitochondria agree with previous results obtained with the perfused lung preparation and indicate that the mitochondria play an important role in the accumulation of basic drugs in the lung.

Impairment in pulmonary bioenergetics following chlorphentermine administration to rats

Biochemical alteration in pulmonary oxidative metabolism and morphological integrity of lung mitochondria were examined in rats following administration of chlorphentermine (30 mg/kg, ip, 5 days per week) for 1 or 2 weeks. During the first week of treatment, body weight gain and food intake were decreased markedly but returned to control levels during the second week. Phospholipid content of the lung was increased 31% and 110% after 1 and 2 weeks of treatment, respectively. This was accompanied by a striking intraalveolar accumulation of hypertrophic alveolar macrophages. The metabolism of both (1- 14C)- and (6- 14C)-glucose was decreased 27% and 26%, respectively, after 2 weeks of drug treatment. In rat lung mitochondria, chlorphentermine significantly lowered the RCR and ADP/O ratio and stimulated state 4 respiration. State 3 respiration and uncoupled state respiration were unaffected. These data indicate that chlorphentermine functions as a true uncoupler of oxidative phosphorylation when administered in vivo. Furthermore, disruption of mitochondrial membranes was observed frequently in lung mitochondria from treated animals. These combined data indicate that the induction of pulmonary phospholipidosis by chlorphentermine is accompanied by marked alterations in subcellular bioenergetics and mitochondrial structure.

The effect of hyperoxia on superoxide production by lung submitochondrial particles

Hyperoxia increases CN −-resistant respiration in rat lung mitochondria, rat lung submitochondrial particles, and porcine lung submitochondrial particles. Cyanide-resistant respiration increases from 0 at normal lung tissue oxygen tensions to 1.2 ± 0.06 nmol O 2 · min −1 · mg protein −1 ( X ± SD ) in rat lung mitochondria and 2.7 ± 0.2 nmol O 2 · min −1 · mg protein −1 in rat lung submitochondrial particles, when assayed at an oxygen concentration of 85%. Superoxide production by submitochondrial particles washed free of superoxide dismutase was directly measured by quantitating superoxide dismutase-inhibitable epinephrine oxidation or cytochrome c reduction, and compared to parallel measurements of CN − resistant respiration. Succinate or NADH was used as substrate and rotenone, antimycin, or CN − were used as respiratory chain inhibitors. At least two components of the respiratory chain produced O 2 −, the ubiquinone-cytochrome b region and the NADH dehydrogenase complex. Superoxide generation by the NADH dehydrogenase is 1.5-fold greater than superoxide production by the ubiquinone-cytochrome b segment. Oxidation of NADH by porcine lung mitochondria, treated with either rotenone, antimycin, or CN −, parallels rates of O 2 − formation and increases directly with oxygen tension, providing another indirect measurement of superoxide production by mitochondrial membranes. These findings support the hypothesis that oxygen toxicity is in part a consequence of increased rates of intracellular O 2 − and H 2O 2 production.

Phospholipid-transfer activity in type II cells isolated from adult rat lung

The soluble fraction from adult rat lung type II cells stimulated the transfer of various phospholipids between either liposomes or rat lung microsomes and rat lung mitochondria. Compared to whole lung, type II cells are highly enriched in transfer activity suggesting that phospholipid-transfer proteins play a role in the transport of surfactant phospholipids. Sephadex chromatography of pH 5.1 supernatant from type II cells yielded only one fraction catalysing the transfer of phosphatidylcholine, phosphatidylglycerol and phosphatidylethanolamine, while chromatography of pH 5.1 supernatant from whole lung yielded in addition a specific phosphatidylcholine-transfer and a specific phosphatidylglycerol-transfer protein.

Phospholipid transfer proteins in rat lung. Identification of a protein specific for phosphatidylglycerol.

From the soluble fraction of a rat lung homogenate, two proteins were partially purified which catalyzed the transfer of phosphatidylglycerol between liposomes and rat lung mitochondria. One protein was specific for phosphatidylglycerol, the other also catalyzed the transfer of phosphatidylcholine and phosphatidylethanolamine. The soluble fraction from rat liver also contained the nonspecific, but not the specific phosphatidylglycerol-transfer protein. It is suggested that the specific phosphatidylglycerol-transfer protein, as well as the phosphatidylcholine-transfer protein, which is also present in rat lung, contribute in the lung to the biogenesis of lamellar bodies, the organelles where surfactant phosphatidylcholine and phosphatidylglycerol are stored prior to secretion onto the alveolar surface.

Sources of carbon for dicarboxylic acids in rat lung mitochondria

In view of previous work which demonstrated negligible pyruvate carboxylase activity, the present experiments were undertaken to investigate the potential roles of glutamate and the purine nucleotide cycle in replenishment of four-carbon units in rat lung mitochondria. 1. 1. Lung mitochondrial preparations were able to form citrate from 2-oxoglutarate, and, in the presence of 2-hydroxymalonate, the addition of L-glutamate resulted in net citrate formation. 2. 2. The rate of oxidation of [l- 14C] glutamate and [l- 14C] 2-oxoglutarate to 14CO 2 measured in these preparations was further increased in uncoupled mitochondria. 3. 3. Significant purine nucleotide cycle activity was observed in mitochondria-free lung tissue preparations. Additionally, significant net accumulation of ammonia, malate and fumarate was observed in the complete system; whereas when aspartate or GTP were omitted, no detectable fumarate and only low levels of ammonia and malate formation were observed. 4. 4. It is concluded that the limited ability to oxidize glutamate and the demonstration of purine nucleotide cycle activity provides at least 2 potential mechanisms for the maintenance of tricarboxylic acid cycle intermediates in lung mitochondria.

Ozone interaction with rodent lung. II. Effects on oxygen consumption of mitochondria.

Abstract In order to examine oxidant effects on lung cellular oxidative metabolism, mitochondrial O 2 consumption (substrate oxidation) was studied in the lungs of O 3 -exposed animals. Male rats (60 to 90-day-old) were exopsed to O 3 either acutely (2 p.p.m. for 8 hours) or subacutely (0.8 p.p.m. for 10 to 20 days). For in vitro experiments suspensions of rat lung homogenates or rat lung mitochondria were exposed to a flow of 10 to 15 p.p.m. O 3 per minute for 20 minutes; both homogenate and mitochondrial preparations absorbed O 3 at a rate of 2.3 to 5.0 p.p.m. per minute (depending upon the amount of tissue). A total of 30 to 40 nmoles O 3 per milligram of protein was absorbed during this period. Subsequent to acute exposures, the rate of O 2 consumption (oxidation of succinate) in lung homogenate or mitochondrial fraction decreased approximately 25 per cent (p 3 exposures, subacute exposures resulted in an augmentation of lung cellular O 2 utilization. As high as 45 per cent (p 2 consumption occurred in homogenates of subacutely exposed rat lungs compared to those of control rat lungs. The specific activity (e.g., succinate oxidase activity per milligram protein) in the isolated mitochondral fraction was only 15 per cent (p