5999
J Physiol 591.23 (2013) pp 5999–6015
Hydrogen sulphide inhibits Ca2+ release through InsP3 receptors and relaxes airway smooth muscle Isabel Castro-Piedras1,2 and Jose F. Perez-Zoghbi1,2 1
Department of Cell Physiology and Molecular Biophysics and 2 Center for Membrane Protein Research, Texas Tech University Health Sciences Center, MS 6551, 3601 4th Street, Lubbock, TX 79430, USA
Key points • The novel signalling molecule hydrogen sulphide (H2 S) regulates diverse cell physiological
processes in several organs and systems including airway smooth muscle contractility.
The Journal of Physiology
• We explored the mechanisms of H2 S-induced smooth muscle relaxation in small intra-
pulmonary airways using lung slices and imaging approaches.
• We found that exogenous and endogenous H2 S inhibited intracellular Ca2+ release specifically
through the inositol-1,4,5-trisphosphate (InsP3 ) receptor in smooth muscle cells and reversibly inhibited acetylcholine-induced intracellular Ca2+ oscillations, thus leading to airway dilatation. • The effects of H2 S on InsP3 -induced Ca2+ release and airway contraction were mimicked by the reducing agent dithiothreitol and inhibited by the oxidizing agent diamide, suggesting that H2 S acts as a thiol-reducing agent to reduce Ca2+ release through InsP3 receptors and to evoke relaxation. • Our results suggest that endogenously produced H2 S is a novel modulator of InsP3 -mediated Ca2+ signalling in airway smooth muscle and thus promotes bronchodilatation.
Abstract Hydrogen sulphide (H2 S) is a signalling molecule that appears to regulate diverse cell physiological process in several organs and systems including vascular and airway smooth muscle cell (SMC) contraction. Decreases in endogenous H2 S synthesis have been associated with the development of cardiovascular diseases and asthma. Here we investigated the mechanism of airway SMC relaxation induced by H2 S in small intrapulmonary airways using mouse lung slices and confocal and phase-contrast video microscopy. Exogenous H2 S donor Na2 S (100 μM) reversibly inhibited Ca2+ release and airway contraction evoked by inositol-1,4,5-trisphosphate (InsP3 ) uncaging in airway SMCs. Similarly, InsP3 -evoked Ca2+ release and contraction was inhibited by endogenous H2 S precursor L-cysteine (10 mM) but not by L-serine (10 mM) or either amino acid in the presence of DL-propargylglycine (PPG). Consistent with the inhibition of Ca2+ release through InsP3 receptors (InsP3 Rs), Na2 S reversibly inhibited acetylcholine (ACh)-induced Ca2+ oscillations in airway SMCs. In addition, Na2 S, the H2 S donor GYY-4137, and L-cysteine caused relaxation of airways pre-contracted with either ACh or 5-hydroxytryptamine (5-HT). Na2 S-induced airway relaxation was resistant to a guanylyl cyclase inhibitor (ODQ) and a protein kinase G inhibitor (Rp-8-pCPT-cGMPS). The effects of H2 S on InsP3 -evoked Ca2+ release and contraction as well as on the relaxation of agonist-contracted airways were mimicked by the thiol-reducing agent dithiothreitol (DTT, 10 mM) and inhibited by the oxidizing agent diamide (30 μM). These studies indicate that H2 S causes airway SMC relaxation by inhibiting Ca2+ release through InsP3 Rs and consequent reduction of agonist-induced Ca2+ oscillations in SMCs. The results suggest a novel role for endogenously produced H2 S that involves the modulation of
C 2013 The Authors. The Journal of Physiology C 2013 The Physiological Society
DOI: 10.1113/jphysiol.2013.257790
6000
I. Castro-Piedras and J. F. Perez-Zoghbi
J Physiol 591.23
InsP3 -evoked Ca2+ release – a cell-signalling system of critical importance for many physiological and pathophysiological processes. (Received 29 April 2013; accepted after revision 15 October 2013; first published online 21 October 2013) Corresponding author J. F. Perez-Zoghbi: Department of Cell Physiology and Molecular Biophysics, Texas Tech University Health Sciences Center, 3601 4th Street, Lubbock, TX 79423, USA. Email: [email protected] Abbreviations ACh, acetylcholine; 2-APB, 2-aminoethoxydiphenyl borate; BCA, β-cyanoalanine; CBS, cystathionine β-synthase; CSE, cystathionine γ-lyase; Cys, L-cysteine; DTT, dithiothreitol; H2 S, hydrogen sulphide; 5-HT, 5-hydroxytryptamine (serotonin); InsP3 , inositol trisphosphate; InsP3 R, InsP3 receptor; ODQ, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one; PKG, protein kinase G; PPG, DL-propargylglycine; Ser, L-serine; SMC, smooth muscle cell.
Introduction Most living organisms produce small amounts of the toxic gas H2 S. Recent studies in mammals suggest that H2 S is a signalling molecule that appears to regulate diverse cell physiological process in several organs and systems including dilatation of blood vessels and modulation of neurotransmission (Yang et al. 2008; Li et al. 2011; Wang, 2012). Endogenous H2 S in mammalian cells is produced mainly from L-cysteine and homocysteine by two pyridoxal-5 -phosphate (vitamin B6 )-dependent enzymes, cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE) and by 3-mercaptopyruvate sulphurtransferase in combination with aminotransferase (Kimura, 2011; Renga, 2011). Alteration of endogenous H2 S production has been linked to pathophysiology process including hypertension and several inflammatory diseases (Yang et al. 2008; Vandiver & Snyder, 2012). All H2 S synthesizing enzymes have been found to be expressed in the lung although there are differences in the presence of individual enzymes between species. Both CSE and CBS were found expressed in primary cultures of human airway SMCs (Perry et al. 2011). In the mouse, CSE and CBS expression is localized in the SMCs of the small intrapulmonary airways as well as in blood vessels but these enzymes are absent in alveolar and airway epithelial cells (Chen et al. 2009; Wang et al. 2011). Importantly, the expression of CSE (but not of CBS) as well as the levels of endogenous H2 S in lung tissue are reduced in both rat and mouse models of allergic asthma (Chen et al. 2009; Zhang et al. 2013). In addition, these studies found that administration of exogenous H2 S to asthmatic mice improved lung function and decreased airway inflammation and remodelling. Furthermore, CSE gene knockout (KO) mice developed stronger airway hyperresponsiveness than the wild-type mice during allergen challenge (Zhang et al. 2013). Finally, serum H2 S levels were found to be decreased in both adult and pediatric asthma patients and they were positively correlated with lung function parameters including forced expiratory volume in 1 s (Wu et al. 2008; Tian et al. 2012).
These findings suggest that the CSE–H2 S system plays a critical protective role in the development of asthma. Contraction of SMCs in the small intrapulmonary airways is fundamental for the development of airway hyperresponsiveness and reversible airway obstruction that characterize asthma and other obstructive lung diseases (Burgel, 2011). According with its effects in vivo, exogenous H2 S (0.3–3 mM) has been shown to cause relaxation of mouse (Jiang et al. 2007; Kubo et al. 2007) and pig (Rashid et al. 2013) small (
J Physiol 591.23 (2013) pp 5999–6015
Hydrogen sulphide inhibits Ca2+ release through InsP3 receptors and relaxes airway smooth muscle Isabel Castro-Piedras1,2 and Jose F. Perez-Zoghbi1,2 1
Department of Cell Physiology and Molecular Biophysics and 2 Center for Membrane Protein Research, Texas Tech University Health Sciences Center, MS 6551, 3601 4th Street, Lubbock, TX 79430, USA
Key points • The novel signalling molecule hydrogen sulphide (H2 S) regulates diverse cell physiological
processes in several organs and systems including airway smooth muscle contractility.
The Journal of Physiology
• We explored the mechanisms of H2 S-induced smooth muscle relaxation in small intra-
pulmonary airways using lung slices and imaging approaches.
• We found that exogenous and endogenous H2 S inhibited intracellular Ca2+ release specifically
through the inositol-1,4,5-trisphosphate (InsP3 ) receptor in smooth muscle cells and reversibly inhibited acetylcholine-induced intracellular Ca2+ oscillations, thus leading to airway dilatation. • The effects of H2 S on InsP3 -induced Ca2+ release and airway contraction were mimicked by the reducing agent dithiothreitol and inhibited by the oxidizing agent diamide, suggesting that H2 S acts as a thiol-reducing agent to reduce Ca2+ release through InsP3 receptors and to evoke relaxation. • Our results suggest that endogenously produced H2 S is a novel modulator of InsP3 -mediated Ca2+ signalling in airway smooth muscle and thus promotes bronchodilatation.
Abstract Hydrogen sulphide (H2 S) is a signalling molecule that appears to regulate diverse cell physiological process in several organs and systems including vascular and airway smooth muscle cell (SMC) contraction. Decreases in endogenous H2 S synthesis have been associated with the development of cardiovascular diseases and asthma. Here we investigated the mechanism of airway SMC relaxation induced by H2 S in small intrapulmonary airways using mouse lung slices and confocal and phase-contrast video microscopy. Exogenous H2 S donor Na2 S (100 μM) reversibly inhibited Ca2+ release and airway contraction evoked by inositol-1,4,5-trisphosphate (InsP3 ) uncaging in airway SMCs. Similarly, InsP3 -evoked Ca2+ release and contraction was inhibited by endogenous H2 S precursor L-cysteine (10 mM) but not by L-serine (10 mM) or either amino acid in the presence of DL-propargylglycine (PPG). Consistent with the inhibition of Ca2+ release through InsP3 receptors (InsP3 Rs), Na2 S reversibly inhibited acetylcholine (ACh)-induced Ca2+ oscillations in airway SMCs. In addition, Na2 S, the H2 S donor GYY-4137, and L-cysteine caused relaxation of airways pre-contracted with either ACh or 5-hydroxytryptamine (5-HT). Na2 S-induced airway relaxation was resistant to a guanylyl cyclase inhibitor (ODQ) and a protein kinase G inhibitor (Rp-8-pCPT-cGMPS). The effects of H2 S on InsP3 -evoked Ca2+ release and contraction as well as on the relaxation of agonist-contracted airways were mimicked by the thiol-reducing agent dithiothreitol (DTT, 10 mM) and inhibited by the oxidizing agent diamide (30 μM). These studies indicate that H2 S causes airway SMC relaxation by inhibiting Ca2+ release through InsP3 Rs and consequent reduction of agonist-induced Ca2+ oscillations in SMCs. The results suggest a novel role for endogenously produced H2 S that involves the modulation of
C 2013 The Authors. The Journal of Physiology C 2013 The Physiological Society
DOI: 10.1113/jphysiol.2013.257790
6000
I. Castro-Piedras and J. F. Perez-Zoghbi
J Physiol 591.23
InsP3 -evoked Ca2+ release – a cell-signalling system of critical importance for many physiological and pathophysiological processes. (Received 29 April 2013; accepted after revision 15 October 2013; first published online 21 October 2013) Corresponding author J. F. Perez-Zoghbi: Department of Cell Physiology and Molecular Biophysics, Texas Tech University Health Sciences Center, 3601 4th Street, Lubbock, TX 79423, USA. Email: [email protected] Abbreviations ACh, acetylcholine; 2-APB, 2-aminoethoxydiphenyl borate; BCA, β-cyanoalanine; CBS, cystathionine β-synthase; CSE, cystathionine γ-lyase; Cys, L-cysteine; DTT, dithiothreitol; H2 S, hydrogen sulphide; 5-HT, 5-hydroxytryptamine (serotonin); InsP3 , inositol trisphosphate; InsP3 R, InsP3 receptor; ODQ, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one; PKG, protein kinase G; PPG, DL-propargylglycine; Ser, L-serine; SMC, smooth muscle cell.
Introduction Most living organisms produce small amounts of the toxic gas H2 S. Recent studies in mammals suggest that H2 S is a signalling molecule that appears to regulate diverse cell physiological process in several organs and systems including dilatation of blood vessels and modulation of neurotransmission (Yang et al. 2008; Li et al. 2011; Wang, 2012). Endogenous H2 S in mammalian cells is produced mainly from L-cysteine and homocysteine by two pyridoxal-5 -phosphate (vitamin B6 )-dependent enzymes, cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE) and by 3-mercaptopyruvate sulphurtransferase in combination with aminotransferase (Kimura, 2011; Renga, 2011). Alteration of endogenous H2 S production has been linked to pathophysiology process including hypertension and several inflammatory diseases (Yang et al. 2008; Vandiver & Snyder, 2012). All H2 S synthesizing enzymes have been found to be expressed in the lung although there are differences in the presence of individual enzymes between species. Both CSE and CBS were found expressed in primary cultures of human airway SMCs (Perry et al. 2011). In the mouse, CSE and CBS expression is localized in the SMCs of the small intrapulmonary airways as well as in blood vessels but these enzymes are absent in alveolar and airway epithelial cells (Chen et al. 2009; Wang et al. 2011). Importantly, the expression of CSE (but not of CBS) as well as the levels of endogenous H2 S in lung tissue are reduced in both rat and mouse models of allergic asthma (Chen et al. 2009; Zhang et al. 2013). In addition, these studies found that administration of exogenous H2 S to asthmatic mice improved lung function and decreased airway inflammation and remodelling. Furthermore, CSE gene knockout (KO) mice developed stronger airway hyperresponsiveness than the wild-type mice during allergen challenge (Zhang et al. 2013). Finally, serum H2 S levels were found to be decreased in both adult and pediatric asthma patients and they were positively correlated with lung function parameters including forced expiratory volume in 1 s (Wu et al. 2008; Tian et al. 2012).
These findings suggest that the CSE–H2 S system plays a critical protective role in the development of asthma. Contraction of SMCs in the small intrapulmonary airways is fundamental for the development of airway hyperresponsiveness and reversible airway obstruction that characterize asthma and other obstructive lung diseases (Burgel, 2011). According with its effects in vivo, exogenous H2 S (0.3–3 mM) has been shown to cause relaxation of mouse (Jiang et al. 2007; Kubo et al. 2007) and pig (Rashid et al. 2013) small (
