Key Words: mitochondria Ⅲ reactive oxygen species Ⅲ ratiometric fluorescent protein sensor Ⅲ hypoxic pulmonary vasoconstriction S mall pulmonary arteries constrict in response to a decrease in alveolar oxygen tension through a physiological response termed hypoxic pulmonary vasoconstriction (HPV). Despite intensive study since the first description of HPV in 1946, the mechanism of O 2 sensing remains unclear. Mitochondria have been considered a putative site of cellular oxygen sensing because they consume O 2 and therefore represent the intracellular site with the lowest oxygen tension. Mitochondria generate superoxide and hydrogen peroxide during cellular respiration, so changes in the production of reactive oxygen species (ROS) during hypoxia could potentially signal changes in O 2 tension and trigger cellular responses. However, whether hypoxia increases or decreases ROS generation during HPV remains unclear.Using chemiluminescence in whole perfused lungs and endothelium-denuded rings of distal pulmonary arteries, Michelakis, Archer, and Weir and colleagues detected decreases in ROS generation during hypoxia. [1][2][3][4] Similarly, in cultured pulmonary arterial pulmonary artery smooth muscle cells (PASMCs), Mehta et al detected a decrease in ROS generation as determined by dihydrodichlorofluorescein diacetate (DCF), dihydroethidium, and Amplex red. 5 By contrast, using cultured PASMCs, we found that hypoxia triggers an increase in ROS generation. 6 -8 Our studies used the ROS-sensitive indicator dichlorofluorescein (DCF), mitochondrial inhibitors, enzymatic and chemical antioxidants and mitochondria-deficient ( 0 ) cells. Other investigators have detected similar increases in ROS generation during hypoxia by using lucigenin-derived chemiluminescence and electron paramagnetic resonance spectrometry in small pulmonary arteries, as well as DCF in PASMCs. 8,9 Attempts to resolve the question of whether ROS levels increase or decrease during hypoxia have been hindered by the technical limitations of the tools used to assess oxidant stress. Although lucigenin can detect ROS, lucigenin itself is capable of producing superoxide in cells. 9 Furthermore,

The use of inhaled nitric oxide in premature infants with the respiratory distress syndrome decreases the incidence of chronic lung disease and death.

Abstract-We hypothesized that mitochondria function as the O 2 sensors underlying hypoxic pulmonary vasoconstriction by releasing reactive oxygen species (ROS) from complex III of the electron transport chain (ETC). We have previously found that antioxidants or inhibition of the proximal region of the ETC attenuates hypoxic pulmonary vasoconstriction in rat lungs and blocks hypoxia-induced contraction of isolated pulmonary arterial (PA) myocytes. Key Words: reactive oxygen species Ⅲ hypoxia Ⅲ redox signaling Ⅲ pulmonary circulation Ⅲ oxidants H ypoxic pulmonary vasoconstriction (HPV) diverts blood flow away from the lung during fetal development and optimizes lung gas exchange after birth by enhancing the matching of blood flow and ventilation. Excised lungs retain the HPV response, 1-6 as do rings of the pulmonary artery (PA) 7,8 even when they are denuded of endothelium. 9,10 Even isolated PA myocytes contract during hypoxia, 11 indicating that an O 2 sensor is intrinsic to those cells.Although HPV has been well characterized, the underlying mechanism of O 2 sensing is not established. Mitochondria have long been known to generate reactive oxygen species (ROS), 12 although these oxidants have classically been viewed as toxic byproducts of the electron transport pathway, possibly contributing to the effects of aging. 13 More recently, mitochondrial ROS have been implicated as intracellular signaling agents. We previously reported that mitochondria increase ROS generation during hypoxia and that site-specific inhibition of electron transport could attenuate hypoxiainduced ROS generation. 14,15 In isolated buffered-perfused rat lungs and isolated PA myocytes, we found that inhibition of the mitochondrial electron transport chain (ETC) upstream from complex III attenuated HPV. 16 By contrast, inhibition downstream from complex III either had no effect or augmented HPV. 16 The likely explanation for these findings is that O 2 -dependent ROS production occurs within complex III. In the Q cycle, a free radical (ubisemiquinone) is normally generated during the electron transport process. This radical can potentially donate its unpaired electron to O 2 , thereby generating superoxide. Our model suggests that the process of ROS generation from that site is amplified during hypoxia.In support of this model, we found that antioxidants selectively abolish the HPV response in isolated lungs. 16 However, that study did not test whether the calcium increases during hypoxia required ROS production. Accordingly, the present study sought to determine whether oxidant production from the mitochondria is responsible for triggering calcium increases and therefore myocyte contraction during HPV. This hypothesis was tested by measuring the effects of site-specific mitochondrial inhibitors and antioxidants on calcium signaling in primary cultured PA myocytes under hypoxic conditions.

Abstract-Mitochondria have been implicated as a potential site of O 2 sensing underlying hypoxic pulmonary vasoconstriction (HPV), but 2 disparate models have been proposed to explain their reaction to hypoxia. One model proposes that hypoxia-induced increases in mitochondrial reactive oxygen species (ROS) generation activate HPV through an oxidant-signaling pathway, whereas the other proposes that HPV is a result of decreased oxidant signaling.In an attempt to resolve this debate, we use a novel, ratiometric, redox-sensitive fluorescence resonance energy transfer (HSP-FRET) probe, in concert with measurements of reduced/oxidized glutathione (GSH/GSSG), to assess cytosolic redox responses in cultured pulmonary artery smooth muscle cells (PASMCs). Superfusion of PASMCs with hypoxic media increases the HSP-FRET ratio and decreases GSH/GSSG, indicating an increase in oxidant stress. The antioxidants pyrrolidinedithiocarbamate and N-acetyl-L-cysteine attenuated this response, as well as the hypoxiainduced increases in cytosolic calcium ([Ca 2ϩ ] i ), assessed by the Ca 2ϩ -sensitive FRET sensor YC2.3. Adenoviral overexpression of glutathione peroxidase or cytosolic or mitochondrial catalase attenuated the hypoxia-induced increase in ROS signaling and [Ca 2ϩ ] i . Adenoviral overexpression of cytosolic Cu, Zn-superoxide dismutase (SOD-I) had no effect on the hypoxia-induced increase in ROS signaling and [Ca 2ϩ ] i , whereas mitochondrial matrix-targeted Mn-SOD (SOD-II) augmented [Ca 2ϩ ] i . The mitochondrial inhibitor myxothiazol attenuated the hypoxia-induced changes in the ROS signaling and [Ca 2ϩ ] i , whereas cyanide augmented the increase in [Ca 2ϩ ] i . Finally, simultaneous measurement of ROS and Ca 2ϩ signaling in the same cell revealed that the initial increase in these 2 signals could not be distinguished temporally. These results demonstrate that hypoxia triggers increases in PASMC [Ca 2ϩ ] i by augmenting ROS signaling from the mitochondria. Key Words: hypoxic pulmonary vasoconstriction Ⅲ reactive oxygen species Ⅲ redox signaling Ⅲ antioxidants Ⅲ fluorescence resonance energy transfer A lthough hypoxic pulmonary vasoconstriction (HPV) was first described by von Euler and Liljestrand in 1946, 1 the underlying mechanism by which vascular cells detect the decrease in O 2 tension has not been established. Hypoxia activates an O 2 sensor that triggers contraction of pulmonary artery smooth muscle cells (PASMCs) through an increase in cytosolic calcium ([Ca 2ϩ ] i ) via release of Ca 2ϩ from the sarcoplasmic reticulum and/or entry of extracellular Ca 2ϩ through voltage-, receptor-, or store-operated channels in the sarcolemma. [2][3][4][5][6][7][8] However, the signaling pathways that couple the O 2 sensor to the increases in [Ca 2ϩ ] i have not been established.Mitochondria have long been considered putative sites of oxygen sensing because they consume O 2 and therefore represent the intracellular site with the lowest oxygen tension.Two opposing views have emerged regarding the nature of the...

Voltage-gated Kv2.1 potassium channels are important in the brain for determining activity-dependent excitability. Small ubiquitin-like modifier proteins (SUMOs) regulate function through reversible, enzyme-mediated conjugation to target lysine(s). Here, sumoylation of Kv2.1 in hippocampal neurons is shown to regulate firing by shifting the half-maximal activation voltage (V1/2) of channels up to 35 mV. Native SUMO and Kv2.1 are shown to interact within and outside channel clusters at the neuronal surface. Studies of single, heterologously expressed Kv2.1 channels show that only K470 is sumoylated. The channels have four subunits, but no more than two non-adjacent subunits carry SUMO concurrently. SUMO on one site shifts V1/2 by 15 mV, whereas sumoylation of two sites produces a full response. Thus, the SUMO pathway regulates neuronal excitability via Kv2.1 in a direct and graded manner.

Small ubiquitin modifier 1 (SUMO1) is shown to regulate K2P1 background channels in the plasma membrane (PM) of live mammalian cells. Confocal microscopy reveals native SUMO1, SAE1, and Ubc9 (the enzymes that activate and conjugate SUMO1) at PM where SUMO1 and expressed human K2P1 are demonstrated to colocalize. Silent K2P1 channels in excised PM patches are activated by SUMO isopeptidase (SENP1) and resilenced by SUMO1. K2P1-Lys274 is crucial: when mutated to Gln, Arg, Glu, Asp, Cys, or Ala, the channels are constitutively active and insensitive to SUMO1 and SENP1. Tandem mass spectrometry confirms conjugation of SUMO1 to the ε-amino group of Lys274 in vitro. FRET microscopy shows that assembly of K2P1 and SUMO1 requires Lys274. Singleparticle TIRF microscopy shows that wild-type channels in PM have two K2P1 subunits and assemble with two SUMO1 monomers. Although channels engineered with one Lys274 site carry just one SUMO1 they are activated and silenced by SENP1 and SUMO1 like wild-type channels.T he activities of many cellular proteins are regulated by covalent conjugation of small ubiquitin modifier (SUMO) proteins on ε-amino groups of specific lysine residues. Such proteins are sumoylated and desumoylated via conserved enzymes that form or hydrolyze isopeptide bonds to these target lysines. This pathway is well-recognized to modulate nuclear import and export, DNA repair, and transcription factor activity (1). Unexpectedly, we found the SUMO pathway to regulate the activity of human K2P1 potassium channels expressed at the PM of Xenopus laevis oocytes and COS-7 African Green Monkey fibroblasts (2). At baseline, K2P1 channels in PM were electrically silent due to the indigenous SUMO machinery. Channel activation required exposure to an isopeptidase (SENP1) that removes SUMO or mutation of a specific lysine residue of K2P1, Lys274. Regulatory events were shown to be reversible by "cramming" of channels in PM patches into cells producing SENP1 or SUMO1. Mutation of Lys274 prevented SUMO conjugation producing active channels insensitive to suppression by SUMO1 or activation by SENP1.Many biochemical and structural studies have examined the interaction of SUMO with transcription factors. In contrast, the mechanisms underlying SUMO regulation at the PM of cells has been controversial in the literature. Even as additional PM substrates for sumoylation were reported [including, Kv2.1 and Kv1.5 voltage-gated potassium channels (3, 4), GluR6 receptors (5), and TRPM4 channels (6)], our description of SUMO action on K2P1 has been questioned (7,8). Here, we confirm the regulation of human K2P1 channels in PM by sumoylation and characterize the SUMO-channel interaction in detail using CHO cells.Results K2P channels are dedicated pathways for background flux of potassium ions that set cellular resting potentials and mediate electrical excitability subject to a broad array of regulatory influences (9). Identified by their unique primary structure of two poreforming domains in each subunit (10) and their operation as potas...

Rationale: The role of reactive oxygen species (ROS) signaling in the O 2 sensing mechanism underlying acute hypoxic pulmonary vasoconstriction (HPV) has been controversial. Although mitochondria are important sources of ROS, studies using chemical inhibitors have yielded conflicting results, whereas cellular models using genetic suppression have precluded in vivo confirmation. Hence, genetic animal models are required to test mechanistic hypotheses. Objectives: We tested whether mitochondrial Complex III is required for the ROS signaling and vasoconstriction responses to acute hypoxia in pulmonary arteries (PA). Methods: A mouse permitting Cre-mediated conditional deletion of the Rieske iron-sulfur protein (RISP) of Complex III was generated. Adenoviral Cre recombinase was used to delete RISP from isolated PA vessels or smooth muscle cells (PASMC). Measurements and Main Results: In PASMC, RISP depletion abolished hypoxia-induced increases in ROS signaling in the mitochondrial intermembrane space and cytosol, and it abrogated hypoxia-induced increases in [Ca 21 ] i . In isolated PA vessels, RISP depletion abolished hypoxia-induced ROS signaling in the cytosol. Breeding the RISP mice with transgenic mice expressing tamoxifen-activated Cre in smooth muscle permitted the depletion of RISP in PASMC in vivo. Precision-cut lung slices from those mice revealed that RISP depletion abolished hypoxia-induced increases in [Ca 21 ] i of the PA. In vivo RISP depletion in smooth muscle attenuated the acute hypoxia-induced increase in right ventricular systolic pressure in anesthetized mice. Conclusions: Acute hypoxia induces superoxide release from Complex III of smooth muscle cells. These oxidant signals diffuse into the cytosol and trigger increases in [Ca 21 ] i that cause acute hypoxic pulmonary vasoconstriction.Keywords: oxygen sensing; Rieske iron-sulfur protein; reactive oxygen species; roGFP; hypoxic pulmonary vasoconstrictionIn the lung, alveolar hypoxia triggers acute constriction of small pulmonary arteries (PA), a response termed hypoxic pulmonary vasoconstriction (HPV). This response is recapitulated in cultured PA smooth muscle cells (PASMC), indicating that the oxygen-sensing mechanism underlying HPV is intrinsic to the PASMC (1-12). Our previous work has implicated increases in reactive oxygen species (ROS) signaling during hypoxia (9,10, 12). Previous studies using mitochondrial inhibitors and mitochondria-deficient (r 0 ) cells suggested that the electron transport chain (ETC) is required for hypoxia-induced ROS signaling in the pulmonary circulation (9, 11-18). We subsequently assessed ROS signaling in hypoxic PASMC using roGFP, a thiol-containing, redox-sensitive reporter (19-23) targeted to compartments within mitochondria or the cytosol (10). Unlike other methods (24-26), this targeted approach permitted the differentiation of hypoxia-induced ROS changes between mitochondrial subcompartments. During hypoxia, increased oxidation was detected in the mitochondrial intermembrane space (IMS) and the cytoso...

The standing outward K+ current (IKso) governs the response of cerebellar granule neurons to natural and medicinal stimuli including volatile anesthetics. In this study, we showed that sumoylation silenced half of IKso at the surface of cerebellar granule neurons because the underlying channels were heterodimeric assemblies of K2P1, a subunit subject to sumoylation, and the two P domain, acid-sensitive K+ (TASK) channel subunits, K2P3 or K2P9. The heteromeric channels comprised the acid-sensitive portion of IKso and mediated its response to halothane. We anticipate that sumoylation also influences sensation and homeostatic mechanisms in mammals through TASK channels formed with K2P1.