Browsing by Author "Kiselar, Janna"

Now showing 1 - 2 of 2
  • Thumbnail Image
    Item
    Somatic cell hemoglobin modulates nitrogen oxide metabolism in the human airway epithelium
    (Springer Nature, 2021-07-29) Marozkina, Nadzeya; Smith, Laura; Zhao, Yi; Zein, Joe; Chmiel, James F.; Kim, Jeeho; Kiselar, Janna; Davis, Michael D.; Cunningham, Rebekah S.; Randell, Scott H.; Gaston, Benjamin; Pediatrics, School of Medicine
    Endothelial hemoglobin (Hb)α regulates endothelial nitric oxide synthase (eNOS) biochemistry. We hypothesized that Hb could also be expressed and biochemically active in the ciliated human airway epithelium. Primary human airway epithelial cells, cultured at air–liquid interface (ALI), were obtained by clinical airway brushings or from explanted lungs. Human airway Hb mRNA data were from publically available databases; or from RT-PCR. Hb proteins were identified by immunoprecipitation, immunoblot, immunohistochemistry, immunofluorescence and liquid chromatography- mass spectrometry. Viral vectors were used to alter Hbβ expression. Heme and nitrogen oxides were measured colorimetrically. Hb mRNA was expressed in human ciliated epithelial cells. Heme proteins (Hbα, β, and δ) were detected in ALI cultures by several methods. Higher levels of airway epithelial Hbβ gene expression were associated with lower FEV1 in asthma. Both Hbβ knockdown and overexpression affected cell morphology. Hbβ and eNOS were apically colocalized. Binding heme with CO decreased extracellular accumulation of nitrogen oxides. Human airway epithelial cells express Hb. Higher levels of Hbβ gene expression were associated with airflow obstruction. Hbβ and eNOS were colocalized in ciliated cells, and heme affected oxidation of the NOS product. Epithelial Hb expression may be relevant to human airways diseases.
  • Thumbnail Image
    Item
    Voltage-gated potassium channel proteins and stereoselective S-nitroso-l-cysteine signaling
    (American Society for Clinical Investigation, 2020-08-13) Gaston, Benjamin; Smith, Laura; Bosch, Jürgen; Seckler, James; Kunze, Diana; Kiselar, Janna; Marozkina, Nadzeya; Hodges, Craig A.; Wintrobe, Patrick; McGee, Kellen; Morozkina, Tatiana S.; Burton, Spencer T.; Lewis, Tristan; Strassmaier, Timothy; Getsy, Paulina; Bates, James N.; Lewis, Stephen J.; Pediatrics, School of Medicine
    S-nitroso-l-cysteine (L-CSNO) behaves as a ligand. Its soluble guanylate cyclase–independent (sGC-independent) effects are stereoselective — that is, not recapitulated by S-nitroso-d-cysteine (D-CSNO) — and are inhibited by chemical congeners. However, candidate L-CSNO receptors have not been identified. Here, we have used 2 complementary affinity chromatography assays — followed by unbiased proteomic analysis — to identify voltage-gated K+ channel (Kv) proteins as binding partners for L-CSNO. Stereoselective L-CSNO–Kv interaction was confirmed structurally and functionally using surface plasmon resonance spectroscopy; hydrogen deuterium exchange; and, in Kv1.1/Kv1.2/Kvβ2-overexpressing cells, patch clamp assays. Remarkably, these sGC-independent L-CSNO effects did not involve S-nitrosylation of Kv proteins. In isolated rat and mouse respiratory control (petrosyl) ganglia, L-CSNO stereoselectively inhibited Kv channel function. Genetic ablation of Kv1.1 prevented this effect. In intact animals, L-CSNO injection at the level of the carotid body dramatically and stereoselectively increased minute ventilation while having no effect on blood pressure; this effect was inhibited by the L-CSNO congener S-methyl-l-cysteine. Kv proteins are physiologically relevant targets of endogenous L-CSNO. This may be a signaling pathway of broad relevance.