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Sci Signal. 2015 Apr 21;8(373):ra37. doi: 10.1126/scisignal.2005846.
Protein kinase G-regulated production of H2S governs oxygen sensing.
Yuan G1,
Vasavda C1,
Peng YJ1,
Makarenko VV1,
Raghuraman G1,
Nanduri J1,
Gadalla MM2,
Semenza GL3,
Kumar GK1,
Snyder SH4,
Prabhakar NR5.
- 1
- Institute for Integrative Physiology and Center for Systems Biology for O2 Sensing, Biological Sciences Division, University of Chicago, Chicago, IL 60637, USA.
- 2
- Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA. Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
- 3
- Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA. Departments of Pediatrics, Medicine, Oncology, Radiation Oncology, and Biological Chemistry; and McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
- 4
- Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA. Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA. Department of Psychiatry and Behavioral Sciences, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
- 5
- Institute for Integrative Physiology and Center for Systems Biology for O2 Sensing, Biological Sciences Division, University of Chicago, Chicago, IL 60637, USA. nanduri@uchicago.edu.
Abstract
Reflexes initiated by the carotid body, the principal O2-sensing organ, are critical for maintaining cardiorespiratory homeostasis during hypoxia. O2 sensing by the carotid body requires carbon monoxide (CO) generation by heme oxygenase-2 (HO-2) and hydrogen sulfide (H2S) synthesis by cystathionine-γ-lyase (CSE). We report that O2 stimulated the generation of CO, but not that of H2S, and required two cysteine residues in the heme regulatory motif (Cys(265) and Cys(282)) of HO-2. CO stimulated protein kinase G (PKG)-dependent phosphorylation of Ser(377) of CSE, inhibiting the production of H2S. Hypoxia decreased the inhibition of CSE by reducing CO generation resulting in increased H2S, which stimulated carotid body neural activity. In carotid bodies from mice lacking HO-2, compensatory increased abundance of nNOS (neuronal nitric oxide synthase) mediated O2 sensing through PKG-dependent regulation of H2S by nitric oxide. These results provide a mechanism for how three gases work in concert in the carotid body to regulate breathing.
Copyright © 2015, American Association for the Advancement of Science.
Fig. 1Effect of O2 on CO and H2S generation in HEK-293 cells expressing HO-2 and CSE alone or together
(A) CO generation as a function of PO2 in the medium in cells transfected with the empty vector and HO-2 vector. (B) Effect of mutating cysteine residues in HO-2 on CO generation. Top: Schematic representation of cysteine residues in the N-terminal and heme regulatory motif of HO-2. Bottom: CO production in cells expressing either wild-type (WT) or mutant (C127A, C265A, C282A, or C265A/C282A) HO-2 in response to normoxia and hypoxia. (C) Rate of CO generation as a function of PO2 in WT and mutant HO-2 (C265A/C282A)–expressing cells. Apparent Km values are derived by iterative curve fitting. (D to F) H2S generation as a function of PO2 in cells transfected with the empty vector or CSE vector (D) or vectors encoding CSE and HO-2 (E) or CSE and mutant HO-2 (C265A/C282A) (F). The graphs in (A) to (F) represent means ± SEM (n = 3 to 5 independent experiments). *P < 0.05; **P < 0.01; n.s., not significant (P > 0.05). See for data on Western blot analysis of HO-2– and CSE-expressing HEK cells.
Sci Signal. 2015;8(373):ra37-ra37.
Fig. 2CO inhibits H2S generation through sGC-dependent cGMP production
(A to D) Effects of siRNA silencing of sGCα1 on the generation of cGMP and H2S in response to either the CO donor CORM-2 (A and B) or coexpression of HO-2 (C and D) in HEK-293 cells expressing CSE. Scr, scrambled RNA. (E and F) Analysis of serine phosphorylation of CSE. Top: representative immunoblot; bottom: densitometric analysis. Effects of siRNA silencing of either sGCα1 or cGMP-dependent PKG II on serine phosphorylation in cells expressing CSE in response to CORM-2 (E) or in CSE/HO-2–coexpressing cells under normoxia or hypoxia or hypoxic cells returned to normoxia. (F) The graphs in (A) to (F) represent means ± SEM (n = 4 to 5 independent experiments). **P < 0.01. See for data on silencing sGC and PKG II by siRNA. I.P., immunoprecipitation.
Sci Signal. 2015;8(373):ra37-ra37.
Fig. 3Inhibition of H2S generation by CO requires phosphorylation of CSE at Ser377
(A) Top: Amino acid residues in CSE required for H2S generation. Bottom: Sequence alignment of CSE across species reveals that Ser377 is evolutionarily conserved (red). Conserved residues are outlined by a solid box. Conservative substitutions are shown in gray, and nonconservative substitutions are outlined by a dashed box. (B and C) Comparison of the serine phosphorylation of exogenously expressed WT and mutant (S377A) CSE in cells treated with the CO donor CORM-2 (B) and in cells coexpressing HO-2 (C). Top: representative immunoblots; bottom: densitometric analysis. (D and E) Effects of CORM-2 (D) and HO-2 expression (E) on H2S generation in cells expressing WT or mutant CSE (S377A). The graphs in (B) to (E) represent means ± SEM (n = 3 to 4 independent experiments). **P < 0.01. See for data on the effect of the phosphomimetic mutation S377E on H2S generation.
Sci Signal. 2015;8(373):ra37-ra37.
Fig. 4PKG-dependent cGMP signaling in the carotid body
(A to D) Effect of the PKG inhibitor 8-pCPT on (A) H2S generation and (B to D) baseline sensory nerve activity of WT and CSE−/− mouse carotid bodies. (E to J) Effect of CORM-2 on hypoxia-evoked (E) H2S generation and (F to J) sensory nerve response of WT and CSE−/− mouse carotid bodies with or without 8-pCPT or the sGC inhibitor ODQ. The insets in the tracings of (B), (C), and (F) to (I) show superimposed action potentials of the sensory nerve fiber from which the integrated carotid body sensory nerve activity [CB activity; impulses per second (imp/s)] was derived. Black horizontal bars marked with “Hx” represent the duration of the hypoxic challenge (n = 3 independent experiments for H2S measurements and n = 6 carotid bodies for each genotype and treatment for sensory nerve activity measurements). **P < 0.01. See for data on YC-1.
Sci Signal. 2015;8(373):ra37-ra37.
Fig. 5Gaseous messenger generation and sensory nerve activity in HO-2–null carotid bodies
(A) Adjacent carotid body sections immunostained for HO-2 and tyrosine hydroxylase (TH), a marker of glomus cells, in WT and HO-2−/− mice. Scale bar, 20 µm. (B) CO generation in carotid bodies of WT and HO-2−/− mice. (C to F) H2S generation (C) and sensory nerve activity (D to F) of the carotid bodies of WT, HO-2−/−, and HO-2−/− + CSE−/− mice. In tracings of (D), the insets present superimposed action potentials of the sensory nerve fiber from which the integrated carotid body sensory nerve activity (CB activity; imp/s) was derived, and black bars marked with “Hx” represent the duration of the hypoxic challenge. Images in (A) are representative of three mice per genotype. The graphs in (B), (C), (E), and (F) represent means ± SEM (n = 3 independent experiments for CO and H2S measurements and n = 6 carotid bodies for each genotype for sensory nerve activity measurements). **P < 0.01.
Sci Signal. 2015;8(373):ra37-ra37.
Fig. 6NO signaling in HO-2–null carotid body
(A) Adjacent carotid body sections immunostained for nNOS and tyrosine hydroxylase (TH), amarker of glomus cells, inWT and HO-2−/− mice. Scale bar, 20 µm. (B to E) Effect of the nNOS inhibitor 7-NI on H2S generation (B) and sensory nerve activity (C to E) of carotid bodies from HO-2−/− and HO-2−/− + CSE−/− mice. In tracings in (C), the insets present superimposed action potentials of the sensory nerve fiber from which the integrated carotid body sensory nerve activity (CB activity; imp/s) was derived, and black bars marked with “Hx” represent the duration of the hypoxic challenge. (F and G) Effect of the NO donor NOC-18 on H2S generation (F) and CSE serine phosphorylation (G) in HEK-293 cells expressing WT or mutant CSE (S377A). Images in (A) are representative of three mice per genotype. The graphs in (B) and (D) to (G) represent means ± SEM (n = 3 for each genotype and treatment for H2S measurements, n = 6 to 8 carotid bodies for each genotype and treatment for sensory nerve activity measurements, and n = 3 independent experiments for H2S measurements and CSE phosphorylation in HEK-293 cells). **P < 0.01.
Sci Signal. 2015;8(373):ra37-ra37.
Fig. 7O2 sensing in the carotid body
Schematic presentation of the signaling pathways associated with the interplay between three gases—O2, CO, and H2S—in glomus cells of the carotid body (CB) and their impact on CB neural activity and breathing. Cys265 and Cys282 are located in the heme regulatory motif of HO-2. Ser377 is the target residue in the putative PKG recognition sequence in CSE.
Sci Signal. 2015;8(373):ra37-ra37.
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