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Hydrogen sulphide inhibits Ca 2+ release through InsP 3 receptors and relaxes airway smooth muscle

2013; Wiley; Volume: 591; Issue: 23 Linguagem: Inglês

10.1113/jphysiol.2013.257790

1469-7793

Isabel Castro‐Piedras, Jose F. Perez‐Zoghbi,

Neuroscience of respiration and sleep

The novel signalling molecule hydrogen sulphide (H2S) regulates diverse cell physiological processes in several organs and systems including airway smooth muscle contractility. We explored the mechanisms of H2S-induced smooth muscle relaxation in small intrapulmonary airways using lung slices and imaging approaches. We found that exogenous and endogenous H2S 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 H2S on InsP3-induced Ca2+ release and airway contraction were mimicked by the reducing agent dithiothreitol and inhibited by the oxidizing agent diamide, suggesting that H2S acts as a thiol-reducing agent to reduce Ca2+ release through InsP3 receptors and to evoke relaxation. Our results suggest that endogenously produced H2S is a novel modulator of InsP3-mediated Ca2+ signalling in airway smooth muscle and thus promotes bronchodilatation. Abstract Hydrogen sulphide (H2S) 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 H2S synthesis have been associated with the development of cardiovascular diseases and asthma. Here we investigated the mechanism of airway SMC relaxation induced by H2S in small intrapulmonary airways using mouse lung slices and confocal and phase-contrast video microscopy. Exogenous H2S donor Na2S (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 H2S 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 (InsP3Rs), Na2S reversibly inhibited acetylcholine (ACh)-induced Ca2+ oscillations in airway SMCs. In addition, Na2S, the H2S donor GYY-4137, and l-cysteine caused relaxation of airways pre-contracted with either ACh or 5-hydroxytryptamine (5-HT). Na2S-induced airway relaxation was resistant to a guanylyl cyclase inhibitor (ODQ) and a protein kinase G inhibitor (Rp-8-pCPT-cGMPS). The effects of H2S 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 H2S causes airway SMC relaxation by inhibiting Ca2+ release through InsP3Rs and consequent reduction of agonist-induced Ca2+ oscillations in SMCs. The results suggest a novel role for endogenously produced H2S that involves the modulation of InsP3-evoked Ca2+ release – a cell-signalling system of critical importance for many physiological and pathophysiological processes. Most living organisms produce small amounts of the toxic gas H2S. Recent studies in mammals suggest that H2S 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 H2S 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 H2S production has been linked to pathophysiology process including hypertension and several inflammatory diseases (Yang et al. 2008; Vandiver & Snyder, 2012). All H2S 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 H2S 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 H2S 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 H2S 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–H2S 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 H2S (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 (<5 mm in diameter) bronchial rings pre-contracted with cholinergic agonists. Furthermore, exposure of bronchial rings to 10 mm l-cysteine increased the synthesis of endogenous H2S and induced airway relaxation (Rashid et al. 2013). These results suggest that H2S-induced relaxation of airway SMCs is, at least in part, responsible for the reduction of airway hyperresponsiveness in vivo after the administration of exogenous H2S and this mechanism could mediate some of the protective effects of this molecule against asthma symptoms. The mechanism of H2S-induced airway SMC relaxation is unknown. Relaxation of vascular SMCs in systemic arterial blood vessels is thought to be mediated by activation of plasma membrane ATP-sensitive K+ (KATP) channels in SMCs (Zhao et al. 2001; Cheng et al. 2004; Liang et al. 2011; Wang, 2011). Activation of K+ channels in vascular SMCs leads to membrane hyperpolarization thereby decreasing Ca2+ influx through voltage-dependent Ca2+ channels and eliciting SMC relaxation. In contrast to vascular SMCs, H2S-induced airway SMC relaxation is resistant to KATP channel inhibitors such as glibenclamide, suggesting that KATP channel-independent mechanisms are responsible for airway relaxation (Jiang et al. 2007; Kubo et al. 2007; Rashid et al. 2013). However, an alternative mechanism for H2S-induced airway SMC relaxation has not been proposed. The contraction of airway SMC in small intrapulmonary airways is regulated by intracellular Ca2+ oscillations that result from cyclic Ca2+ release from the SR through the InsP3Rs (Sanderson et al. 2010). Stimuli that evoke airway contraction such as acetylcholine (ACh) and 5-hydroxytryptamine (5-HT) induce InsP3R-mediated Ca2+ oscillations in SMCs (Perez & Sanderson, 2005; Perez-Zoghbi & Sanderson, 2007) whereas stimuli that evoke airway relaxation such as β2-adrenergic receptor agonists and nitric oxide (NO) inhibit these Ca2+ oscillations (Perez-Zoghbi et al. 2010). Furthermore, both β2-adrenergic agonists and NO inhibit InsP3-evoked Ca2+ release, indicating that reduction of Ca2+ oscillations is mediated by inhibition of Ca2+ release through InsP3Rs (Bai & Sanderson, 2006a; Perez-Zoghbi et al. 2010). Whether H2S affects intracellular Ca2+ release through InsP3Rs (or ryanodine receptors) is unknown. However, a recent study shows that long term exposure of HeLa cells to H2S donors up-regulates the expression of InsP3R1 and InsP3R2 but not InsP3R3 (Lencesova et al. 2013). The objective of this work was to investigate the mechanism of H2S-induced SMC relaxation in small intrapulmonary airways. We hypothesized that H2S inhibits Ca2+ release through InsP3Rs and found that exogenous and endogenously-synthesized H2S inhibited InsP3-evoked Ca2+ release, reduced the frequency and amplitude of agonist-induced Ca2+ oscillations, and induced airway relaxation. Furthermore, these H2S effects were mimicked by another thiol-reducing agent and antagonized by an oxidizing agent suggesting that reversible reduction of InsP3R may be involved in the effects of H2S. Our results suggest that endogenous H2S may be a novel regulator of the InsP3R-mediated Ca2+ release and thus it regulates airway SMC contraction and potentially other physiological processes dependent on Ca2+ signalling via InsP3Rs in different cells. Sodium sulphide nonahydrate (Na2S.9H2O), l-cysteine, l-serine, dl-propargylglycine (PPG), acetylcholine (ACh), 5-hydroxytryptamine (5-HT), 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), and caffeine were purchased from Sigma-Aldrich (St Louis, MO, USA). β-Cyano-l-alanine (BCA) and GYY-4137 were from Cayman Chemical, Ann Arbor, MI, USA). Membrane permeable caged-InsP3 (caged iso-inositol(1,4,5)P3/PM) and Rp-8-pCPT-cGMPS were from Enzo Life Sciences (Farmingdale, NY, USA). Ryanodine was from Abcam Biochemicals (Cambridge, MA, USA). 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) was from EMD-Millipore (USA). Hanks' balanced salt solution was supplemented with 20 mm Hepes buffer and adjusted to pH 7.4 (sHBSS). The H2S source salt sodium sulphide (Na2S) was prepared fresh each experimental day, as a 0.1 m stock solution (50 ml), by dissolving the salt in sHBSS and adjusting the pH to 7.40 by slowly adding 12 n HCl. The Na2S fully dissociates and solvates in sHBSS rendering a mixture of ∼75% HS− and ∼25% H2S at 25°C and pH 7.4 (Broderius & Smith, 1977). This Na2S stock solution was maintained in a closed syringe to avoid loss of H2S gas and was diluted to the final concentration approximately 2 min before initiation of each experiment. Solutions were not gasified during the course of the experiments to reduce H2S loss. In all experiments, the lung slices were continuously superfused with sHBSS or the indicated solution in a semi-closed microscope chamber at room temperature. We used Ellman's Reagent (DTNB) to measure spectrophotometrically the concentration of H2S in fresh and discomposed Na2S solutions and in solutions containing Na2S and diamide, as employed in other reports (Li et al. 2008). Aliquots of 20 μl were removed from the Na2S solutions and added to each well of a 96-well microplate containing DTNB (1 mm, 50 μl) and Hepes buffer (pH 8.0, 1 mm, 50 μl). Absorbance at 412 nm was measured with a microplate reader (Tecan, Infinity 200 PRO, Männedorf, Switzerland), subtracted from the blank, and used to calculate the H2S concentration using the Lambert-Beer equation. A reference calibration curve was generated from freshly prepared Na2S solutions in sHBSS (1 μm to 1 mm) to calculate the DTNB molar extinction coefficient (ɛ) in our conditions. The obtained value ɛ= 14,897 m−1 cm−1 was similar to reported DTNB ɛ values (Riddles et al. 1979) and this curve was used to calculate the H2S concentration in all Na2S solutions. Mouse lung slices were prepared as described previously (Mukherjee et al. 2013). Briefly, male C3H mice 8–12 weeks old (Charles River Breeding Labs, MA, USA) were killed with i.p. sodium pentobarbital (40 mg kg−1). The chest cavity was opened and the lungs inflated with ∼1.3 ml of 2% agarose (low-melting temperature agarose, Affymetrix, Santa Clara, CA, USA) in sHBSS at 37°C and ∼0.2 ml of air. The agarose in the lungs was gelled by placing the mouse at 4° C for 20 min. Lungs and heart were removed from the animal, and the lung lobes were cut in 140-μm-thick slices using a tissue slicer (Compresstom VF-300, Precisionary Instruments, San Jose, CA, USA) in a safety cabinet under sterile conditions. Lung slices containing small terminal airways were collected in sHBSS and then incubated in low-glucose Dulbecco's Modified Eagle's Medium (DMEM, Life Technologies, Grand Island, NY, USA) supplemented with antibiotics, at 37°C and 10% CO2 in a cell culture incubator. Lung slices were incubated between 10 and 60 h; no significant changes in airway contraction in response to 100 nm ACh were detected during this period. Lung slices selected for the experiments contained airways with a lumen diameter of 100–250 μm, completely lined by active ciliated epithelial cells, and fully attached to the surrounding lung parenchyma. The Texas Tech University Health Sciences Center Institutional Animal Care & Use Committee approved our animal studies (IACUC no. 07069). The contractile response of airways in lung slices was measured using a phase-contrast video microscopy setup as previously described (Mukherjee et al. 2013). Briefly, lung slices were mounted in a cover-glass in a custom-made perfusion chamber and held in place with a small sheet of nylon mesh. Perfusion of the lung slices was performed by adding solution at one end of the chamber and removing it by suction at the opposite end by means of a custom-made, gravity-fed, computer-controlled perfusion system consisting of eight syringe tubes connected to individual electronic solenoid valves (Lee Company, Westbrook, CT, USA) and to an 8-way manifold. The lung slices were continuously superfused with one of the solutions at ∼800 μl min−1 and solution changes were made by switching between solutions at pre-programmed times using the electronic valves. Exposure of lung slices to two or more drugs/chemicals (e.g. ACh + Na2S) was made by superfusing a single solution containing all drugs/chemicals at the final concentration. The chamber was placed on the stage of an inverted phase-contrast microscope and lung slices were imaged with a 10× objective. Digital images were recorded to a hard drive in time-lapse (0.5 Hz) using a CCD camera, frame grabber, and image acquisition software (Video Savant, IO Industries, Ontario, Canada). The airway cross-sectional luminal area (lumen area) was calculated from each image using a custom-written script in Video Savant that distinguishes the airway lumen from the surrounding tissue. The lumen area was normalized to the initial area (before stimulation) and the changes in lumen area were plotted versus time using graphics software. Approximately 10–12 lung slices were incubated for 50 min at 30°C in 2 ml of sHBSS supplemented with 20 μm Oregon Green 488 BAPTA-1 acetoxymethyl ester (Life technologies, Grand Island, NY, USA) that was dissolved in 20 μl dry DMSO plus 5 μl 20% Pluronic F-127 (Sigma-Aldrich) in DMSO. Subsequently, the slices were transferred to 2 ml sHBSS and incubated for 50 min at 30°C to allow de-esterification of the acetoxymethyl group. Lung slices were mounted in the perfusion chamber as previously described and fluorescence imaging was performed using a custom-made video-rate confocal microscope (Sanderson & Parker, 2003). The sample was illuminated with a 488 nm laser beam and the fluorescence emission (510–530 nm) was collected with a photomultiplier tube (PMT R3896, Hamamatsu) and frame grabber (Alta-AN, BitFlow). Images were recorded at 15 Hz using Video Savant. The changes in fluorescence intensity were analysed by selecting regions of interest (ROI) ranging from 25 to 49 pixels2. Average fluorescence intensities of an ROI were obtained, frame-by-frame, using a custom-written script that allowed the tracking of the ROI within a SMC as it moved with contraction. Final fluorescence values were expressed as a fluorescence ratio (F/F0) normalized to the initial fluorescence (F0). Line-scan analysis of images was performed by extracting a line of pixels from each image and placing them sequentially to form a time sequence in a single image. Flash photolysis of caged-InsP3 was used to experimentally increase the intracellular InsP3 concentration ([InsP3]i.) Lung slices were incubated with 2 μm caged-InsP3-PM (Enzo Life Sciences) for 45 min at 30°C in sHBSS containing 0.1% Pluronic F-127 followed by de-esterification for 30 min in sHBSS. A flash of UV light was produced from a mercury arc lamp, filtered with a band-pass filter (330 nm), and focused into the sample using a biconvex lens (focal distance 200 mm) on the confocal microscope or using the manufacturer's fluorescence adapter (Nikon) in the phase-contrast microscope. The illumination was adjusted to uniformly irradiate a circular region on the sample using an iris diaphragm at a conjugate image plane in the microscope. Flash duration and intensity were controlled using a Uniblitz shutter (Vincent Associates, Rochester, NY, USA) with electronic timing control and neutral density filters, respectively. For determination of changes in [Ca2+]i associated with an InsP3 photorelease, the lung slices were initially loaded with Oregon Green 488 BAPTA-1 AM as described above and then with caged-InsP3-PM. To clamp the [Ca2+]i, airway SMCs were made permeable to external Ca2+ without using detergents or toxins. This approach does not damage the cell membrane and exploits the inherent Ca2+-permeable ion channels of the cells. Lung slices were exposed to 20 mm caffeine and 25 μm ryanodine for 4 min, followed by a washout with sHBSS for 10 min. This treatment locks the ryanodine receptors (RyRs) in the sarcoplasmic reticulum (SR) of airway SMCs in an open state and consequently depletes the intracellular Ca2+ stores. This, in turn, increases Ca2+ influx via store-operated channels across the plasma membrane. Full details and validation for this technique have been reported (Bai & Sanderson, 2006b; Perez-Zoghbi & Sanderson, 2007). In caffeine–ryanodine treated lung slices, the [Ca2+]i of airway SMCs depends on the extracellular Ca2+ concentration ([Ca2+]e). By exposing the lung slices to sHBSS containing 1.3 mm Ca2+, the [Ca2+]i was clamped at a sustained level above the basal (Oregon Green fluorescence F/F0 was 1.4–1.7). In these experiments, the [Ca2+]i was examined by confocal microscopy and was confirmed not to change during the addition of ACh or H2S. Statistical values are expressed as means ± standard error of the mean (SEM). Student's t test or one-way analysis of variance (ANOVA) followed by Tukey's comparison was used to evaluate the significance between means from two or more groups, respectively. Supplemental Figs S1–S10 show supporting results referred in the Results section. Supplemental Videos 1–4, consisting of sequences of confocal fluorescence or phase-contrast images, were produced with 'Video Savant'. Video 1 shows InsP3-evoked Ca2+ waves in absence and presence of Na2S. Supplemental Video 2 shows airway contraction stimulated by caffeine and by InsP3 photorelease. Video 3 shows Ca2+ oscillations in airway SMCs induced by ACh and inhibited by Na2S. Video 4 shows airway contraction induced by ACh and relaxation induced by Na2S in a lung slice. To test the effects of exogenous H2S (derived from Na2S; see Methods) on Ca2+ release from intracellular Ca2+ stores via InsP3Rs in small airway SMCs we loaded mouse lung slices with caged InsP3 and fluorescent [Ca2+]i indicator and measured the [Ca2+]i changes in airway SMCs in response to the photolytic release of intracellular InsP3 (Fig. 1A). In the absence of Na2S, a brief and localized ultraviolet (UV) light flash in a limited region within a SMC evoked a Ca2+ transient that propagated along the longitudinal axes of the SMC as a Ca2+ wave (Fig. 1B) or triggered a few Ca2+ waves (Supplemental Video 1). This indicates that InsP3 photorelease in airway SMCs activated Ca2+ release from intracellular Ca2+ stores through InsP3Rs, as previously demonstrated (Perez-Zoghbi et al. 2010). Subsequent superfusion of the lung slices with 100 μm Na2S had no effects on the basal [Ca2+]i; however, it strongly inhibited the Ca2+ wave evoked by an identical UV flash (Fig. 1B). The subsequent removal of Na2S by superfusing sHBSS restored the ability of a third UV flash to evoke a Ca2+ wave of similar amplitude to the first UV flash. Inhibition of the InsP3-evoked Ca2+ wave was also observed when the membrane-permeable InsP3R inhibitor 2-aminoethoxydiphenyl borate (2-APB) was used instead of Na2S. These results suggest that exogenous H2S reversibly inhibits intracellular Ca2+ release through InsP3Rs in small airway SMCs. Exogenous and endogenous H2S inhibit InsP3-evoked Ca2+ release in airway SMCs A, fluorescence image of part of an airway in a lung slice showing a SMC and epithelial cells (EPCs) that line the airway lumen. The black-edged circle indicates the illumination area with the 30 ms UV flashes, the black-edged square indicates a region of interest (ROI) used to determine the Ca2+ transients (traces in B and C) and the dashed white line indicates the row of pixels along the SMC selected to make the line-scan analysis (images below traces in B and C) to show the Ca2+ waves propagation. B, the effect of 100 μm Na2S (exogenous H2S) and 100 μm 2-APB (labels above traces) on Ca2+ transients (traces) and Ca2+ waves (images below traces) evoked by UV flashes (arrows) to uncage InsP3. Ca2+ transients and waves evoked by InsP3 were inhibited by Na2S (n= 12 from 8 mice) and by 2-APB (n= 4 from 2 mice) and the inhibition was reversed after Na2S and 2-APB washout (bars graph). C, the effect 10 mm l-cysteine (Cys, precursor of endogenous H2S) and 10 mm l-serine (Ser) on Ca2+ transients and Ca2+ waves evoked by UV flashes. InsP3-evoked Ca2+ transients were inhibited by l-cysteine but not by l-serine or by these amino acids in the presence of 10 mm PPG (n= 5–7 from 4 mice, bars graph). Error bars denote SEM, NS, not significant; **P < 0.01; ***P < 0.001; paired t test. The UV flash-evoked Ca2+ waves in an airway SMC before, during, and after Na2S exposure is shown in Supplemental Video 1. Endogenous H2S is synthesized in the SMC cytoplasm mainly from l-cysteine in a reaction catalysed by cystathionine γ-lyase (CSE; Yang et al. 2008; Li et al. 2011; Fu et al. 2012). Addition of l-cysteine to several SMC preparations including airways increases the production of H2S four- to sixfold (Cheng et al. 2004; d'Emmanuele di Villa Bianca et al. 2009; Rashid et al. 2013). Mouse small airways express CSE in the SMCs but not in the epithelial cells or in the surrounding lung parenchyma (Wang et al. 2011). In addition, the amino acid transport system alanine–serine–cysteine is highly active in SMCs (Jiang et al. 2007). Therefore, we studied whether addition of l-cysteine to the sHBSS affects InsP3-evoked Ca2+ release in airway SMCs. For these experiments we used lung slices maintained in serum-free culture medium for ∼48 h because CSE expression and activity in SMCs has been shown to increase under these conditions (Yang et al. 2011). Figure 1C shows that l-cysteine, but not l-serine, significantly inhibited the amplitude of the InsP3-evoked Ca2+ waves. Furthermore, the effect of l-cysteine was blocked by the CSE inhibitor dl-propargylglycine (PPG, 10 mm) even though this inhibitor did not affect InsP3-evoked Ca2+ wave in the presence of l-serine (Fig. 1C, bar plot). These results suggest that H2S generated in the SMCs from l-cysteine by CSE, similarly to exogenous H2S, inhibits Ca2+ release through InsP3R. Airway SMCs contract in response to InsP3-evoked Ca2+ waves (Perez-Zoghbi et al. 2010). Therefore we determined whether H2S affects the airway contraction stimulated by InsP3 photorelease. We loaded lung slices with caged-InsP3 and assessed the changes in airway cross-sectional luminal area (lumen area hereafter) in response to an UV flash that illuminated an area that overlaps the whole airway (Fig. 2A). Since caffeine activates Ca2+ release from ER/SR Ca2+ stores through ryanodine receptors (RyRs) in airway SMCs and evokes transient contraction (Perez & Sanderson, 2005), we initially stimulated lung slices with a caffeine pulse to test airway contractility and to normalize the UV flash-evoked response. Both caffeine and the UV flash evoked airway SMC contraction and transiently reduced airway lumen area (Fig. 2B and Supplemental Video 2). Unloaded lung slices did not contract in response to a 2.0 s UV flash (Supplemental Fig. S1A and D), whereas UV flash-evoked airway contraction in caged-InsP3-loaded slices increased with the UV flash duration between 0.3 and 2.0 s (Supplemental Fig. S1B–D), suggesting that UV flash-evoked airway contraction depends on the amount of InsP3 photoreleased. In the presence of Na2S, the UV flash-evoked airway contraction was inhibited for UV flashes lasting between 0.3 and 2 s (Fig. 2B and Supplemental Fig. S1B–D). Similarly, 2-APB reduced the UV flash-evoked airway contraction confirming that this contraction depends on InsP3R activation (Fig. 2B). In addition, the UV flash-evoked airway contraction was inhibited by l-cysteine but not by l-serine or either amino acid in the presence of PPG (Fig. 2C) suggesting that InsP3-evoked airway contraction was inhibited by the H2S synthesized endogenously from l-cysteine by CSE. Inhibition of InsP3-evoked Ca2+ release and contraction by H2S may in principle occur as a result of H2S-induced depletion of the Ca2+ stores. However, H2S did not affect caffeine-induced airway contraction (Supplemental Fig. S2), suggesting that H2S neither depletes the ER/SR Ca2+ stores nor inhibits the Ca2+ release through RyRs. Together, these results show that H2S specifically inhibits InsP3R-mediated Ca2+ release and thus inhibits airway contraction. Exogenous and endogenous H2S inhibit InsP3-evoked airway contraction A, phase-contrast image shows an airway in a lung slice and a dashed line delimits the area illuminated by the UV flash to uncage InsP3. B, the effect of 100 μm Na2S (middle trace) and 100 μm 2-APB (lower trace) on airway contraction evoked by a UV flash (arrows) to uncage InsP3. Airway contraction was initially stimulated by 20 mm caffeine (Caff) and subsequently by a 1.5 s UV flash; both stimuli induced contraction and transiently reduced airway lumen area. UV flash-evoked airway contractions were inhibited by Na2S and by 2-APB (n= 6–9 from 6 mice, bars graph). C, the effect 10 mm l-cysteine (Cys) and 10 mm l-serine (Ser) on airway contraction evoked by UV flashes. InsP3-evoked airway contraction was inhibited by l-cysteine but not l-serine or by either amino acid in the presence of PPG (n= 6 from 4 mice, bars graph). Error bars denote SEM, **P < 0.01, ***P < 0.001, one-way analysis of variance followed by Tukey's comparison. The airway contraction stimulated by caffeine and subsequently by an UV flash is shown in Supplemental Video 2. Stimulation of airways with agonists that are ligands of the Gq/11 protein-coupled receptors (e.g. ACh) induces Ca2+ oscillations that are mediated by cyclic Ca2+ release through the InsP3Rs (Perez-Zoghbi et al. 2009). We investigated whether exogenous H2S inhibits such Ca2+ oscillations. Figure 3 and Supplemental Video 3 show that the addition of Na2S decreased the frequency and amplitude (fluorescence intensity) of the ACh-evoked Ca2+ oscillations and the integrated SMC [Ca2+]i signal. These changes were closely followed by an increase in airway lumen area, suggesting that the Na2S-induced inhibition of Ca2+ oscillations leads to SMC relaxation. Subsequent Na2S washout in the continuous presence of ACh partially restored the high frequency, amplitude, and integral of the Ca2+ oscillations and decreased the airway lumen area, indicating that Na2S effects were reversible. H2S inhibits ACh-induced Ca2+ oscillations in airway SMCs A, the effect of 100 μm Na2S on the Ca2+ oscillations induced by 100 nm ACh in an airway SMCs (top trace), the integral of these Ca2+ oscillations (middle trace), and the airway lumen area (bottom trace). B, line-scan analysis from the longitudinal axes of a SMC at the times indicated by numbered bars under the top trace in A showing the effect of Na2S on the Ca2+ waves (vertical white lines) and its reversibility after H2S washout. C, summary of the effects of Na2S addition and washout on the frequency and amplitude of ACh-induced Ca2+ oscillations. Error bars denote SEM, n= 6 from 4 mice, **P < 0.01, ***P < 0.001, paired t test. The Ca2+ oscillations induced by ACh, its inhibition by Na2S and reversibility after Na2S washout of the representative experiment presented here are shown in Supplemental Video 3. Because intracellular Ca2+ release during Ca2+ oscillations activates Ca2+ entry via plasma membrane store-operated Ca2+ channels and this Ca2+ entry is necessary to maintain the Ca2+ oscillations, we tested if H2S affects the Ca2+ entry via this mechanism. We used caffeine to activate Ca2+ store depletion via RyR activation, independently of InsP3R activation. Caffeine induced a [Ca2+]i transient increase followed by a [Ca2+]i sustained increase in airway SMC (Supplemental Fig. S3) indicative of Ca2+ release and Ca2+ influx, respectively (Perez & Sanderson, 2005). The [Ca2+]i sustained increase was reversibly reduced by extracellular Ca2+ removal (Supplemental Fig. S3A), but not by Na2S (Supplemental Fig. S3B), i.e. Na2S did not block Ca2+ entry from the extracellular compartment. These results suggest that exogenous H2S reversibly inhibits agonist-induced Ca2+ oscillations by inhibiting the Ca2+ release through the InsP3Rs but not by inhibiting extracellular Ca2+ entry via the store-operated Ca2+ channels. We next characterized the effects of H2S on agonist-induced airway contraction. Airways were initially stimulated with ACh which produced a sustained airway contraction as previously described (Perez & Sanderson, 2005). Subsequent addi