Hyperoxia Causes Mitochondrial Fragmentation in Pulmonary Endothelial Cells by Increasing Expression of Pro-Fission Proteins

Objective—We explored mechanisms that alter mitochondrial structure and function in pulmonary endothelial cells (PEC) function after hyperoxia. Approach and Results—Mitochondrial structures of PECs exposed to hyperoxia or normoxia were visualized and mitochondrial fragmentation quantified. Expression of pro-fission or fusion proteins or autophagy-related proteins were assessed by Western blot. Mitochondrial oxidative state was determined using mito-roGFP. Tetramethylrhodamine methyl ester estimated mitochondrial polarization in treatment groups. The role of mitochondrially derived reactive oxygen species in mt-fragmentation was investigated with mito-TEMPOL and mitochondrial DNA (mtDNA) damage studied by using ENDO III (mt-tat-endonuclease III), a protein that repairs mDNA damage. Drp-1 (dynamin-related protein 1) was overexpressed or silenced to test the role of this protein in cell survival or transwell resistance. Hyperoxia increased fragmentation of PEC mitochondria in a time-dependent manner through 48 hours of exposure. Hyperoxic PECs exhibited increased phosphorylation of Drp-1 (serine 616), decreases in Mfn1 (mitofusion protein 1), but increases in OPA-1 (optic atrophy 1). Pro-autophagy proteins p62 (LC3 adapter–binding protein SQSTM1/p62), PINK-1 (PTEN-induced putative kinase 1), and LC3B (microtubule-associated protein 1A/1B-light chain 3) were increased. Returning cells to normoxia for 24 hours reversed the increased mt-fragmentation and changes in expression of pro-fission proteins. Hyperoxia-induced changes in mitochondrial structure or cell survival were mitigated by antioxidants mito-TEMPOL, Drp-1 silencing, or inhibition or protection by the mitochondrial endonuclease ENDO III. Hyperoxia induced oxidation and mitochondrial depolarization and impaired transwell resistance. Decrease in resistance was mitigated by mito-TEMPOL or ENDO III and reproduced by overexpression of Drp-1. Conclusions—Because hyperoxia evoked mt-fragmentation, cell survival and transwell resistance are prevented by ENDO III and mito-TEMPOL and Drp-1 silencing, and these data link hyperoxia-induced mt-DNA damage, Drp-1 expression, mt-fragmentation, and PEC dysfunction

Redox signaling via oxidative inactivation of PTEN modulates pressure-dependent myogenic tone in rat middle cerebral arteries.

The present study examined the level of generation of reactive oxygen species (ROS) and roles of inactivation of the phosphatase PTEN and the PI3K/Akt signaling pathway in response to an increase in intramural pressure-induced myogenic cerebral arterial constriction. Step increases in intraluminal pressure of cannulated cerebral arteries induced myogenic constriction and concomitant formation of superoxide (O2 (.-)) and its dismutation product hydrogen peroxide (H2O2) as determined by fluorescent HPLC analysis, microscopic analysis of intensity of dihydroethidium fluorescence and attenuation of pressure-induced myogenic constriction by pretreatment with the ROS scavenger 4,hydroxyl-2,2,6,6-tetramethylpiperidine1-oxyl (tempol) or Mito-tempol or MitoQ in the presence or absence of PEG-catalase. An increase in intraluminal pressure induced oxidation of PTEN and activation of Akt. Pharmacological inhibition of endogenous PTEN activity potentiated pressure-dependent myogenic constriction and caused a reduction in NPo of a 238 pS arterial KCa channel current and an increase in [Ca(2+)]i level in freshly isolated cerebral arterial muscle cells (CAMCs), responses that were attenuated by Inhibition of the PI3K/Akt pathway. These findings demonstrate an increase in intraluminal pressure induced increase in ROS production triggered redox-sensitive signaling mechanism emanating from the cross-talk between oxidative inactivation of PTEN and activation of the PI3K/Akt signaling pathway that involves in the regulation of pressure-dependent myogenic cerebral arterial constriction

Peripheral antinociceptive effects of a bifunctional μ and δ opioid receptor ligand in rat model of inflammatory bladder pain.

There is a need to develop a novel analgesic for pain associated with interstitial cystitis/painful bladder syndrome (IC/PBS). The use of the conventional μ-opioid receptor agonists to manage IC/PBS pain is controversial due to adverse CNS effects. These effects are attenuated in benzylideneoxymorphone (BOM), a low-efficacy μ-opioid receptor agonist/δ-opioid receptor antagonist that attenuates thermal pain and is devoid of reinforcing effects. We hypothesize that BOM will inhibit bladder pain by attenuating responses of urinary bladder distension (UBD)-sensitive afferent fibers. Therefore, the effect of BOM was tested on responses of UBD-sensitive afferent fibers in L6 dorsal root from inflamed and non-inflamed bladder of rats. Immunohistochemical (IHC) examination reveals that following the induction of inflammation there were significant high expressions of μ, δ, and μ-δ heteromer receptors in DRG. BOM dose-dependently (1-10 mg/kg, i.v) attenuated mechanotransduction properties of these afferent fibers from inflamed but not from non-inflamed rats. In behavioral model of bladder pain, BOM significantly attenuated visceromotor responses (VMRs) to UBD only in inflamed group of rats when injected either systemically (10 mg/kg, i.v.) or locally into the bladder (0.1 ml of 10 mg/ml). Furthermore, oxymorphone (OXM), a high-efficacy μ-opioid receptor agonist, attenuated responses of mechanosensitive bladder afferent fibers and VMRs to UBD. Naloxone (10 mg/kg, i.v.) significantly reversed the inhibitory effects of BOM and OXM on responses of bladder afferent fibers and VMRs suggesting μ-opioid receptor-related analgesic effects of these compounds. The results reveal that a low-efficacy, bifunctional opioid-based compound can produce analgesia by attenuating mechanotransduction functions of afferent fibers innervating the urinary bladder

Effect of increasing intraluminal pressure in cannulated cerebral arterial segments on the activity levels of the phosphatase PTEN and the kinase Akt.

<p>Pressurization of the cannulated cerebral arterial segments at 120 mm Hg for 60 minutes exhibited a significant oxidative inactivation of PTEN, whereas this increase in intraluminal pressure induced elevated level of phosphorylated Akt as revealed by the change in density of the expression level of the protein bands for PTEN and phospho-Akt. Bar graphs depict summary of the density of the protein bands presented as mean ± SEM for the oxidized and reduced forms of PTEN and for that of phospho-Akt. n = 2–3 trials per group. *P<0.01, ^# P<0.05 compared to control group.</p

Schematic depicting conceptual presentation of consequences of an increase in intraluminal pressure-induced production of the ROS O₂^– and H₂O₂ on cellular signaling events.

<p>The increase in intraluminal pressure-induced O₂^– and H₂O₂ production triggers a redox sensitive signaling event via oxidative inactivation of the lipid phosphatase PTEN that induces activation of PI3K resulting in generation of phosphatidylinositol (PtdIns) (3,4,5,)P3 (PIP3) and release of IP3 and activation and recruitment of Akt, which could cause phosphorylation and inhibition of K_Ca channel activity, membrane depolarization and activation of L-type Ca²⁺ channel (L-I_Ca), which together with the released IP3 increase intracellular Ca²⁺ level required to evoke pressure-dependent cerebral arterial myogenic constriction.</p

Fluorescence HPLC determination of the level of formation of O₂^– in response to an increase in intraluminal pressure.

<p>The elevation in intraluminal pressure from 40 mm Hg to 120 mm Hg induced a significantly increased concentration of 2-OH-E⁺, the oxidation product of the superoxide detecting probe DHE by O₂^–, normalized to tissue protein (n = 4 separate experiments for each group, *denote P<0.05 compared to control level of 2-OH-E⁺ to that following the increase in intraluminal pressure to 120 mm Hg.</p

Dismutation of the ROS superoxide and H₂O₂ attenuated the increase in intraluminal pressure-induced myogenic constriction of cerebral arterial segments.

<p>Increasing intraluminal pressure over the range of 60 mm Hg to 120 mm Hg in steps of 20 mm Hg induced pressure-dependent myogenic cerebral arterial constriction that was significantly attenuated and converted to vasodilation by pretreatment of the cannulated pressurized cerebral arterial segments with the superoxide dismutase mimic tempol and tempol plus the H₂O₂ dismutase PEG-catalase (A) or with mitochondrial targeted mito-tempol and mito-tempol plus PEG-catalase (B). Data are presented as mean value ± SEM, n = 6–8 cerebral arterial segments per group. *P<0.05, **P<0.001.</p