Jeremy D. Marks

Mitochondrial Reactive Oxygen Species Trigger Calcium Increases During Hypoxia in Pulmonary Arterial Myocytes

Abstract— We hypothesized that mitochondria function as the O2 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. To determine whether the hypoxia-induced increases in mitochondrial ROS act to trigger calcium increases, we measured changes in cytosolic calcium ([Ca2+]i) using fura 2-AM (fluorescence at 340/380 nm) during perfusion with hypoxic media (Po2 12 mm Hg). Hypoxia caused an increase in fura 2 fluorescence, indicating an increase in [Ca2+]i. In superfused PA myocytes, diphenyleneiodonium, rotenone, and myxothiazol, which inhibit the proximal region of the ETC, attenuated hypoxia-induced calcium increases. Antimycin A and cyanide, which inhibit the distal region of the ETC, failed to abolish hypoxia-induced [Ca2+]i increases. To test whether mitochondrial H2O2 is required to trigger [Ca2+]i increases, catalase was overexpressed in PA myocytes with the use of a recombinant adenovirus. Catalase overexpression attenuated hypoxia-induced increases in [Ca2+]i, suggesting that H2O2 acts upstream from calcium increases during hypoxia. These results support the conclusion that mitochondria function as O2 sensors during hypoxia and demonstrate that ROS generated in the proximal region of the ETC act as second messengers to trigger calcium increases in PA myocytes during acute hypoxia.

Hypoxia Triggers Subcellular Compartmental Redox Signaling in Vascular Smooth Muscle Cells

Rationale: Recent studies have implicated mitochondrial reactive oxygen species (ROS) in regulating hypoxic pulmonary vasoconstriction (HPV), but controversy exists regarding whether hypoxia increases or decreases ROS generation. Objective: This study tested the hypothesis that hypoxia induces redox changes that differ among subcellular compartments in pulmonary (PASMCs) and systemic (SASMCs) smooth muscle cells. Methods and Results: We used a novel, redox-sensitive, ratiometric fluorescent protein sensor (RoGFP) to assess the effects of hypoxia on redox signaling in cultured PASMCs and SASMCs. Using genetic targeting sequences, RoGFP was expressed in the cytosol (Cyto-RoGFP), the mitochondrial matrix (Mito-RoGFP), or the mitochondrial intermembrane space (IMS-RoGFP), allowing assessment of oxidant signaling in distinct intracellular compartments. Superfusion of PASMCs or SASMCs with hypoxic media increased oxidation of both Cyto-RoGFP and IMS-RoGFP. However, hypoxia decreased oxidation of Mito-RoGFP in both cell types. The hypoxia-induced oxidation of Cyto-RoGFP was attenuated through the overexpression of cytosolic catalase in PASMCs. Conclusions: These results indicate that hypoxia causes a decrease in nonspecific ROS generation in the matrix compartment, whereas it increases regulated ROS production in the IMS, which diffuses to the cytosol of both PASMCs and SASMCs.

Increases in Mitochondrial Reactive Oxygen Species Trigger Hypoxia-Induced Calcium Responses in Pulmonary Artery Smooth Muscle Cells

Mitochondria have been implicated as a potential site of O2 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 hypoxia-induced increases in cytosolic calcium ([Ca2+]i), assessed by the Ca2+-sensitive FRET sensor YC2.3. Adenoviral overexpression of glutathione peroxidase or cytosolic or mitochondrial catalase attenuated the hypoxia-induced increase in ROS signaling and [Ca2+]i. Adenoviral overexpression of cytosolic Cu, Zn-superoxide dismutase (SOD-I) had no effect on the hypoxia-induced increase in ROS signaling and [Ca2+]i, whereas mitochondrial matrix–targeted Mn-SOD (SOD-II) augmented [Ca2+]i. The mitochondrial inhibitor myxothiazol attenuated the hypoxia-induced changes in the ROS signaling and [Ca2+]i, whereas cyanide augmented the increase in [Ca2+]i. Finally, simultaneous measurement of ROS and Ca2+ 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 [Ca2+]i by augmenting ROS signaling from the mitochondria.

Oxidant Stress during Simulated Ischemia Primes Cardiomyocytes for Cell Death during Reperfusion

Ischemia-reperfusion injury induces oxidant stress, and the burst of reactive oxygen species (ROS) production after reperfusion of ischemic myocardium is sufficient to induce cell death. Mitochondrial oxidant production may begin during ischemia prior to reperfusion because reducing equivalents accumulate and promote superoxide production. We utilized a ratiometric redox-sensitive protein sensor (heat shock protein 33 fluorescence resonance energy transfer (HSP-FRET)) to assess oxidant stress in cardiomyocytes during simulated ischemia. HSP-FRET consists of the cyan and yellow fluorescent protein fluorophores linked by the cysteine-containing regulatory domain from bacterial HSP-33. During ischemia, ROS-mediated oxidation of HSP-FRET was observed, along with a decrease in cellular reduced glutathione levels. These findings were corroborated by measurements using redox-sensitive green fluorescent protein, another protein thiol ratiometric sensor, which became 93% oxidized by the end of simulated ischemia. However, cell death did not occur during ischemia, indicating that this oxidant stress is not sufficient to induce death before reperfusion. However, interventions that attenuate ischemic oxidant stress, including antioxidants or scavengers of residual O2 that attenuate/prevent ROS generation during ischemia, abrogated cell death during simulated reperfusion. These findings reveal that, in isolated cardiomyocytes, sublethal H2O2 generation during simulated ischemia regulates cell death during simulated reperfusion, which is mediated by the reperfusion oxidant burst.

SUMO modification of cell surface Kv2.1 potassium channels regulates the activity of rat hippocampal neurons

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.

Vulnerability of CA1 neurons to glutamate is developmentally regulated

Although it is well documented that glutamate receptor subtypes are differentially expressed during central nervous system development postnatally, how glutamate affects neurons during postnatal development is unclear. We therefore examined the development of the intrinsic neuronal response to glutamate receptor activation by studying single, hippocampal CA1 neurons that had been acutely dissociated from newborn (P1-3), 1 week old (P6-8), and 3 week old (P21-25) rats. Using laser scanning confocal microscopy and the calcium dye Fluo-3, we made time-lapse studies of the effects of glutamate stimulation on free intracellular calcium ([Ca2+]i) and simultaneous changes in neuronal morphology. In P21-25 neurons, glutamate increased [Ca2+]i fluorescence, and caused marked somal swelling, blebbing, and retraction of dendrites into the soma. These major morphological changes were followed by sudden loss of intracellular fluorescence, indicative of a loss of membrane integrity and cell death. In P6-8 neurons, glutamate increased [Ca2+]i to the same extent, but this increase was not followed by either major morphological changes or loss of membrane integrity. In P1-3 neurons, glutamate increased [Ca2+]i minimally, and no morphologic changes were observed. P1-3 neurons dissociated without enzymatic digestion demonstrated glutamate responses identical to responses seen in neurons dissociated with enzymatic digestion. In the presence of MK-801 (15 microM), glutamate still increased [Ca2+]i and caused cell death in P21-25 neurons, but the latency to these effects more than tripled. This late, MK-801-resistant [Ca2+]i increase was not eliminated by DNQX or Ni2+/Cd2+, suggesting that this increase is mediated by metabotropic receptors. These findings demonstrate that (1) hippocampal neurons from newborns are intrinsically less vulnerable to glutamate toxicity than neurons from 3 weeks old animals, and (2) multiple glutamate receptor subtypes affect the magnitude of the [Ca2+]i increase in response to glutamate in the neuronal microenvironment.

Superoxide Generated at Mitochondrial Complex III Triggers Acute Responses to Hypoxia in the Pulmonary Circulation

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(2+)](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(2+)](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(2+)](i) that cause acute hypoxic pulmonary vasoconstriction.

CLC-3 Channels Modulate Excitatory Synaptic Transmission in Hippocampal Neurons

It is well established that ligand-gated chloride flux across the plasma membrane modulates neuronal excitability. We find that a voltage-dependent Cl(-) conductance increases neuronal excitability in immature rodents as well, enhancing the time course of NMDA receptor-mediated miniature excitatory postsynaptic potentials (mEPSPs). This Cl(-) conductance is activated by CaMKII, is electrophysiologically identical to the CaMKII-activated CLC-3 conductance in nonneuronal cells, and is absent in clc-3(-/-) mice. Systematically decreasing [Cl(-)](i) to mimic postnatal [Cl(-)](i) regulation progressively decreases the amplitude and decay time constant of spontaneous mEPSPs. This Cl(-)-dependent change in synaptic strength is absent in clc-3(-/-) mice. Using surface biotinylation, immunohistochemistry, electron microscopy, and coimmunoprecipitation studies, we find that CLC-3 channels are localized on the plasma membrane, at postsynaptic sites, and in association with NMDA receptors. This is the first demonstration that a voltage-dependent chloride conductance modulates neuronal excitability. By increasing postsynaptic potentials in a Cl(-) dependent fashion, CLC-3 channels regulate neuronal excitability postsynaptically in immature neurons.

Mitochondrial Nitric Oxide Mediates Decreased Vulnerability of Hippocampal Neurons from Immature Animals to NMDA

Mitochondrial membrane potential (ΔΨm)-dependent Ca2+ uptake plays a central role in neurodegeneration after NMDA receptor activation. NMDA-induced ΔΨm dissipation increases during postnatal development, coincident with increasing vulnerability to NMDA. NMDA receptor activation also produces nitric oxide (NO), which can inhibit mitochondrial respiration, dissipating ΔΨm. Because ΔΨm dissipation reduces mitochondrial Ca2+ uptake, we hypothesized that NO mediates the NMDA-induced ΔΨm dissipation in immature neurons, underlying their decreased vulnerability to excitotoxicity. Using hippocampal neurons cultured from 5- and 19-d-old rats, we measured NMDA-induced changes in [Ca2+]cytosol, ΔΨm, NO, and [Ca2+]mito. In postnatal day 5 (P5) neurons, NMDA mildly dissipated ΔΨm in a NO synthase (NOS)-dependent manner and increased NO. The NMDA-induced NO increase was abolished with carbonyl cyanide 4-(trifluoromethoxy)phenyl-hydrazone and regulated by [Ca2+]mito. Mitochondrial Ca2+ uptake inhibition prevented the NO increase, whereas inhibition of mitochondrial Ca2+ extrusion increased it. Consistent with this mitochondrial regulation, NOS and cytochrome oxidase immunoreactivity demonstrated mitochondrial localization of NOS. Furthermore, NOS blockade increased mitochondrial Ca2+ uptake during NMDA. Finally, at physiologic O2 tensions (3% O2), NMDA had little effect on survival of P5 neurons, but NOS blockade during NMDA markedly worsened survival, demonstrating marked neuroprotection by mitochondrial NO. In P19 neurons, NMDA dissipated ΔΨm in an NO-insensitive manner. NMDA-induced NO production was not regulated by ΔΨm, and NOS immunoreactivity was cytosolic, without mitochondrial localization. NOS blockade also protected P19 neurons from NMDA. These data demonstrate that mitochondrial NOS mediates much of the decreased vulnerability to NMDA in immature hippocampal neurons and that cytosolic NOS contributes to NMDA toxicity in mature neurons.

One SUMO is sufficient to silence the dimeric potassium channel K2P1

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. Single-particle 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.