Thomas J. Barna

Cytokine Activation of Nuclear Factor κB in Vascular Smooth Muscle Cells Requires Signaling Endosomes Containing Nox1 and ClC-3

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 &kgr;B in vascular smooth muscle cells (SMCs). Tumor necrosis factor-&agr; and interleukin (IL)-1&bgr; stimulation of SMCs resulted in diphenylene iodonium-sensitive ROS production within intracellular vesicles. Nox1 and p22phox, 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-&agr; and interleukin-1&bgr; generation of ROS. Cytokine activation of nuclear factor &kgr;B in SMCs required both Nox1 and ClC-3. We conclude that in response to tumor necrosis factor-&agr; and interleukin-1&bgr;, 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.

Chloride ion currents contribute functionally to norepinephrine-induced vascular contraction

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.

Altered GABAergic function accompanies hippocampal degeneration in mice lacking ClC-3 voltage-gated chloride channels

Mice lacking ClC-3 chloride channels, encoded by the Clcn3 gene, undergo neurodegeneration of the hippocampal formation and retina [Neuron, 29 (2001) 185-196; Genes Cells, 7 (2002) 597-605]. We independently created a mouse lacking the Clcn3 gene which demonstrated similar central nervous system abnormalities, including early postnatal degeneration of retinal photoreceptors. However, we observed a characteristic spatial-temporal sequence of hippocampal neurodegeneration that differs from the pattern previously reported. Anterior-to-posterior degeneration and astrogliosis of the dentate gyrus and hippocampus progressed over months. Sequential loss of hippocampal neuronal subpopulations began in the dentate gyrus and progressed to CA3, followed by CA1 neurons. Projection neurons of the entorhinal cortex degenerated, secondary to the loss of their synaptic targets within the hippocampal formation. Other characteristics of the Clcn3(-/-) mice included an abnormal gait, kyphosis, and absence of hindlimb escape extension upon tail elevation. Spontaneous seizures were observed in four adult Clcn3(-/-) mice, and one mouse died during the event. We hypothesized that neuronal injury may be related to recurrent seizures. Clcn3(-/-) mice had normal serum electrolytes and pH, and exhibited neither hyperglycemia nor rebound hypoglycemia following a glucose load. They displayed a greatly reduced susceptibility to pentylenetetrazole-induced seizures and an abnormally prolonged sedation to benzodiazepines. There was no change in vulnerability to kainic acid-induced seizures. Immunostaining revealed a progressive loss of GABA synthesizing cells in the dentate gyrus. The death of these cells was preceded by increased GABA(A) receptor immunoreactivity. These data suggest that GABA(A) inhibitory neurotransmission is altered in Clcn3(-/-) mice. The increase in GABA(A) receptor density may represent a compensatory response either to chronic excessive excitatory stimuli or reduced inhibitory input from local GABAergic interneurons within the dentate gyrus.

Responses of Fetal Ovine Systemic and Umbilical Arteries to Angiotensin II

Angiotensin II (ANG II) contracts umbilical arteries and has been hypothesized to regulate fetal blood pressure primarily by altering umbilical vascular resistance. To determine whether systemic arteries in term fetal sheep are sensitive to ANG II, isometric contraction of endothelium-intact isolated fetal renal, mesenteric, and umbilical arteries in response to ANG II was studied. ANG II (10−7 M) elicited contractile responses in all three vessels (43 ± 8%, 99 ± 21%, and 105 ± 5% of the maximal response seen with 90 mM KCl for renal, mesenteric, and umbilical arteries, respectively). The time course of the contractile responses differed among the vessels: renal and mesenteric arteries exhibited rapid transient contraction followed by relaxation, whereas umbilical artery displayed a more slowly developing but sustained contraction (1 ± 0%, 3 ± 1%,and 93 ± 4% of maximal contractile response at 5 min, for renal, mesenteric, and umbilical arteries, respectively). The AT1 receptor antagonist, losartan (10−6 M), abolished contractile responses in renal and mesenteric arteries but only slowed the contraction in umbilical artery in response to ANG II and had no effect on maximal tension. AT2 receptor blockade (PD 123319, 10−7 M) had no significant effect on the response to ANG II in any vessel. Indomethacin (10−6 M) significantly potentiated contraction to ANG II in renal and mesenteric but not umbilical arteries. Northern and Western blot analyses demonstrated the presence of AT1 mRNA and protein in all three vessels. Immunostaining for the AT1 receptor was present in endothelium and the tunica media. These findings demonstrate the AT1 receptor is present and functionally active in fetal systemic arteries and are consistent with previous findings that the umbilical circulation displays a greater responsiveness to ANG II than the systemic vasculature.

Complex RNA processing of TDRKH, a novel gene encoding the putative RNA-binding tudor and KH domains.

The sequence from a human EST (IMAGE:259322) with homology to the nucleotide-sensitive chloride conductance regulator (ICln) was used to screen a human aortic cDNA library. The probe sequence was from a region of the EST lacking homology to ICln, and the goal was to isolate an ICln-like gene. A 2843bp cDNA clone with an open reading frame coding for a 561 amino acid protein was isolated. This clone had no homology to ICln. PROSITE analysis of the putative protein sequence reveals one tudor and two K homology (KH) domains. The gene has therefore been named TDRKH. Both KH and tudor motifs are involved in binding to RNA or single-strand DNA. PCR analysis demonstrated that TDRKH is alternatively spliced in several ways and alternatively polyadenylated at multiple sites. Northern analysis confirmed the presence of messages of multiple lengths with predominant bands at 2.8 and 4.0 kb and also demonstrated that TDRKH is widely expressed in human tissues. Within an intron of TDRKH, there is a region with 90% homology to ICln. This sequence, which is incorporated into the alternatively spliced message represented by IMAGE:259322, contains a 2 bp deletion that disrupts the ICln reading frame and therefore represents an ICln pseudogene. The TDRKH gene was mapped to the Epidermal Differentiation Complex (EDC) at chromosome 1q21 by radiation hybrid mapping and STS content of genomic clones from that region. The EDC contains a large cluster of related genes involved in terminal differentiation of the epidermis. It remains to be determined whether TDRKH has a specific role in epithelial function.

51 CLC-3 IS REQUIRED FOR NADPH OXIDASE-DEPENDENT NUCLEAR FACTOR κB ACTIVATION BY SIGNALING ENDOSOMES.

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. Here we show that activation of NADPH oxidase by inflammatory cytokines generates ROS within early endosomes and requires ClC-3, a member of the chloride channel (ClC) family. Nox1, one of multiple membrane-bound catalytic subunits of NADPH oxidase, colocalizes with ClC-3 in early endosomes. Both Nox1 and ClC-3 are necessary for tumor necrosis factor α and interleukin-1β generation of ROS and subsequent activation of the transcription factor NF-κB. We propose that ClC-3 functions as a chloride-proton exchanger and thereby influences ROS production via charge neutralization of the electron flow generated by Nox1 in the endosome. These findings identify ClC-3 as a critical component of the signaling endosome and a novel intermediate in redox-dependent control of gene expression.

The Angiotensin AT 1 Receptor Mediates Vasoconstriction in Fetal Umbilical and Systemic Arteries

The Angiotensin AT 1 Receptor Mediates Vasoconstriction in Fetal Umbilical and Systemic Arteries