Research ArticleCell Biology

Protein kinase G–regulated production of H2S governs oxygen sensing

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Science Signaling  21 Apr 2015:
Vol. 8, Issue 373, pp. ra37
DOI: 10.1126/scisignal.2005846
  • Fig. 1 Effect 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 fig. S1 for data on Western blot analysis of HO-2– and CSE-expressing HEK cells.

  • Fig. 2 CO 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 fig. S3 for data on silencing sGC and PKG II by siRNA. I.P., immunoprecipitation.

  • Fig. 3 Inhibition 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 fig. S3 for data on the effect of the phosphomimetic mutation S377E on H2S generation.

  • Fig. 4 PKG-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 fig. S4 for data on YC-1.

  • Fig. 5 Gaseous 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.

  • Fig. 6 NO signaling in HO-2–null carotid body.

    (A) Adjacent carotid body sections immunostained for nNOS and tyrosine hydroxylase (TH), a marker of glomus cells, in WT 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.

  • Fig. 7 O2 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.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/8/373/ra37/DC1

    Fig. S1. Western blot analysis of HEK-293 cells exogenously expressing HO-2 and CSE.

    Fig. S2. Calcium-dependent calmodulin activity is not required for inhibition of CSE by CO.

    Fig. S3. Effect of siRNA targeting sGCα1 on hypoxia-induced changes in cGMP concentrations and H2S generation in HEK-293 cells.

    Fig. S4. Effects of YC-1 with or without either PKG or sGC inhibitor on H2S generation and the sensory nerve activity of wild-type and CSE−/− mouse carotid bodies.

  • Supplementary Materials for:

    Protein kinase G–regulated production of H2S governs oxygen sensing

    Guoxiang Yuan, Chirag Vasavda, Ying-Jie Peng, Vladislav V. Makarenko, Gayatri Raghuraman, Jayasri Nanduri, Moataz M. Gadalla, Gregg L. Semenza, Ganesh K. Kumar, Solomon H. Snyder, Nanduri R. Prabhakar*

    *Corresponding author. E-mail: nanduri{at}uchicago.edu

    This PDF file includes:

    • Fig. S1. Western blot analysis of HEK-293 cells exogenously expressing HO-2 and CSE.
    • Fig. S2. Calcium-dependent calmodulin activity is not required for inhibition of CSE by CO.
    • Fig. S3. Effect of siRNA targeting sGCα1 on hypoxia-induced changes in cGMP concentrations and H2S generation in HEK-293 cells.
    • Fig. S4. Effects of YC-1 with or without either PKG or sGC inhibitor on H2S generation and the sensory nerve activity of wild-type and CSE−/− mouse carotid bodies.

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    Citation: G. Yuan, C. Vasavda, Y.-J. Peng, V. V. Makarenko, G. Raghuraman, J. Nanduri, M. M. Gadalla, G. L. Semenza, G. K. Kumar, S. H. Snyder, N. R. Prabhakar, Protein kinase G–regulated production of H2S governs oxygen sensing. Sci. Signal. 8, ra37 (2015).

    © 2015 American Association for the Advancement of Science

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