Abstract-Reactive oxygen species (ROS) are mediators of intracellular signals for a myriad of normal and pathologic cellular events, including differentiation, hypertrophy, proliferation, and apoptosis. NADPH oxidases are important sources of ROS that are present in diverse tissues throughout the body and activate many redox-sensitive signal transduction and gene expression pathways. To avoid toxicity and provide specificity of signaling, ROS production and metabolism necessitate tight regulation that likely includes subcellular compartmentalization. However, the constituent elements of NADPH oxidase-dependent cell signaling are not known. To address this issue, we examined cytokine generation of ROS and subsequent activation of the transcription factor nuclear factor B in vascular smooth muscle cells (SMCs). Tumor necrosis factor-␣ and interleukin (IL)-1␤ stimulation of SMCs resulted in diphenylene iodonium-sensitive ROS production within intracellular vesicles. Nox1 and p22 phox , integral membrane subunits of NADPH oxidase, coimmunoprecipitated with early endosomal markers in SMCs. ClC-3, an anion transporter that is primarily found in intracellular vesicles, also colocalized with Nox1 in early endosomes and was necessary for tumor necrosis factor-␣ and interleukin-1␤ generation of ROS. Cytokine activation of nuclear factor B in SMCs required both Nox1 and ClC-3. We conclude that in response to tumor necrosis factor-␣ and interleukin-1␤, NADPH oxidase generates ROS within early endosomes and that Nox1 cannot produce sufficient ROS for cell signaling in the absence of ClC-3. These data best support a model whereby ClC-3 is required for charge neutralization of the electron flow generated by Nox1 across the membrane of signaling endosomes. Key Words: smooth muscle cells Ⅲ NAPDH oxidase Ⅲ cell signaling Ⅲ ion channels I n response to diverse extracellular stimuli, intracellular signaling is dependent on generation of reactive oxygen species (ROS) by NADPH oxidase. The catalytic core of NADPH oxidase consists of a membrane-bound flavocytochrome b558 composed of a Nox (NADPH oxidase) (reviewed elsewhere 1 ) subunit and p22phox . The prototypical model of NAPDH oxidase is found in phagosomes, where the orientation and biochemical properties of this heterodimer obligate reduction of oxygen to superoxide on the side of the membrane opposite from where NADPH and the cytosolic subunits of the oxidase bind. 2 Based on this orientation, activation of NADPH oxidase in nonphagocytes should generate superoxide into the extracellular space or, following endocytosis, into intracellular vesicles. We hypothesized that extracellular stimuli activating the Nox1-based NADPH oxidase would produce superoxide in endocytotic vesicles.The phagocyte NADPH oxidase is electrogenic, moving electrons from cytoplasmic NADPH through the enzyme into the phagosome to reduce oxygen to superoxide. Without charge compensation, this electron flux rapidly depolarizes the membrane (the voltage in the cytoplasm becomes positive relative to the phagoso...

NADPH oxidase activity, phagocytosis, and cell migration are essential functions of polymorphonuclear leukocytes (PMNs) in host defense. The cytoskeletal reorganization necessary to perform these functions has been extensively studied, but the role of cell volume regulation, which is likely dependent upon anion channels, has not been defined. Mice lacking the anion channel ClC-3 (Clcn3 (؊/؊) ) died from presumed sepsis following intravascular catheter placement, whereas Clcn3 (؉/؉) littermates survived. We hypothesized that ClC-3 has a critical role in host defense and reasoned that PMN function would be compromised in these mice. Clcn3(؊/؊) PMNs displayed markedly reduced NADPH oxidase activity in response to opsonized zymosan and modestly reduced activity after phorbol 12-myristate 13-acetate. Human PMNs treated with the anion channel inhibitors niflumic acid or 5-nitro-2-(3-phenylpropylamino)benzoic acid had a very similar defect. ClC-3 protein was detected in the secretory vesicles and secondary granules of resting PMNs and was up-regulated to the phagosomal membrane. Clcn3 (؊/؊) PMNs and human PMNs lacking normal anion channel function both exhibited reduced uptake of opsonized zymosan at 1, 5, and 10 min in a synchronized phagocytosis assay. Niflumic acidtreated PMNs also had impaired transendothelial migration in vitro, whereas migration in vivo was not altered in Clcn3 (؊/؊) PMNs. Selective inhibition of the swelling-activated chloride channel with tamoxifen profoundly reduced PMN migration but had no effect on NADPH oxidase activity. In summary, PMNs lacking normal anion channel function exhibited reduced NADPH oxidase activity, diminished phagocytosis, and impaired migration. ClC-3 was specifically involved in the respiratory burst and phagocytosis.

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Matsuda JJ, Filali MS, Volk KA, Collins MM, Moreland JG, Lamb FS. Overexpression of CLC-3 in HEK293T cells yields novel currents that are pH dependent.

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.

ClC-3, a member of the large superfamily of ClC voltage-dependent Cl -channels, has been proposed as a molecular candidate responsible for volume-sensitive osmolyte and anion channels (VSOACs) in some cells, including heart and vascular smooth muscle. However, the reported presence of native VSOACs in at least two cell types from transgenic ClC-3 disrupted (Clcn3 −/− ) mice casts considerable doubt on this proposed role for ClC-3. We compared several properties of native VSOACs and examined mRNA transcripts and membrane protein expression profiles in cardiac and pulmonary arterial smooth muscle cells from Clcn3 mice, quantitative analysis of ClC mRNA expression levels revealed significant increases in transcripts for ClC-1, ClC-2, and ClC-3, and protein expression profiles obtained using two-dimensional polyacrylamide gel electrophoresis revealed complex changes in at least 35 different unidentified membrane proteins in cells from Clcn3 −/− mice. These findings emphasize that caution needs to be exercised in simple attempts to interpret the phenotypic consequences of conventional global Clcn3 gene inactivation.

Internalization of activated receptors to a compartment enriched with NAPDH oxidase and associated signaling molecules is expected to facilitate regulation of redox-mediated signal transduction. The aim of this study was to test the hypothesis that endocytosis is necessary for generation of reactive oxygen species (ROS) by Nox1 and for redox-dependent signaling in smooth muscle cells (SMCs). Within minutes of treatment with tumor necrosis factor (TNF)-a or thrombin, SMCs increased cellular levels of ROS that was inhibited by shRNA to Nox1. Treatment of SMC with TNF-a induced a dynamin-dependent endosomal generation of ROS, whereas thrombinmediated ROS production did not occur within endosomes and was not prevented by dominant-negative dynamin (dn-dynamin), but instead required transactivation of the epidermal growth factor receptor (EGFR). Activation of the phosphatidylinositol 3-kinase (PI3K)-Akt-activating transcription factor-1 (ATF-1) pathway by TNF-a and thrombin were both Nox1-and dynamin-dependent. In conclusion, we show that formation of specific ligand-receptor complexes results in spatially distinct mechanisms of Nox1 activation and generation of ROS. These findings provide novel insights into the role of compartmentalization for integrating redoxdependent cell signaling. Antioxid. Redox Signal. 12, 583-593.

Norepinephrine (NE) increases Cl− efflux from vascular smooth muscle (VSM) cells. An increase in Cl− conductance produces membrane depolarization. We hypothesized that if Cl− currents are important for agonist-induced depolarization, then interfering with cellular Cl− handling should alter contractility. Isometric contraction of rat aortic rings was studied in a bicarbonate buffer. Substitution of extracellular Cl− with 130 mM methanesulfonate (MS; 8 mM Cl−) did not cause contraction. NE- and serotonin-induced contractions were potentiated in this low-Cl− buffer, whereas responses to K+, BAY K 8644, or NE in the absence of Ca2+ were unaltered. Substitution of Cl− with I− or Br− suppressed responses to NE. Inhibition of Cl−transport with bumetanide (10−5 M) or bicarbonate-free conditions (10 mM HEPES) inhibited NE- but not KCl-induced contraction. The Cl−-channel blockers DIDS (10−3 M), anthracene-9-carboxylic acid (10−3 M), and niflumic acid (10−5 M) all inhibited NE-induced contraction, whereas tamoxifen (10−5 M) did not. Finally, disruption of sarcoplasmic reticular function with cyclopiazonic acid (10−7 M) or ryanodine (10−5 M) prevented the increase in the peak response to NE produced by low-Cl− buffer. We conclude that a Cl− current with a permeability sequence of I−> Br− > Cl− > MS is critical to agonist-induced contraction of VSM.