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Role of protein kinase C and reactive oxygen species in hydrogen sulfide mediated contraction of rat small pulmonary arteries
Abstracts/Journal of Functional Foods 47 (2015) S14–S60
project P20664 and doctoral program BioToP W1224) and COST actions BM 1005 are greatly acknowledged. http://dx.doi.org/10.1016/j.niox.2015.02.140
PP111 Role of protein kinase C and reactive oxygen species in hydrogen sulfide mediated contraction of rat small pulmonary arteries Philip I. Aaronson, Jesus Prieto-Lloret, Yasin Shaifta, Vladimir Snetkov Division of Asthma Allergy & Lung Biology, King’s College London, London, UK The gas H2S (hereafter ‘sulfide’) acts as a vasodilator in the systemic circulation, but elicits a biphasic contraction in pulmonary arteries (PA) via an unknown mechanism. This may be a physiologically important effect; it has also been proposed that the sulfide concentration in PA increases during hypoxia, acting as an oxygen sensor which triggers hypoxic pulmonary vasoconstriction. We studied the mechanism of the sulfide-induced contraction in rings of rat 2nd order PA, using Na2S as a source of sulfide, by recording of isometric force development, the mitochondrial membrane potential (Ψ) using TMRE, and NAD(P)H autofluorescence, and also measured reactive oxygen species (ROS) levels in cultured PA smooth muscle cells using the luminescent ROS indicator LO12 (10 μM). Na2S (1 mM) applied in the presence of 5 μM PGF2a evoked a complex PA contraction comprising an initial transient contraction followed by a relaxation, and then a second and larger contraction which gradually relaxed. Both contractions persisted in the presence of the eNOS blocker L-NAME (300 μM), but were strongly antagonized by the protein kinase C blocker Gö6983 (3 μM) and by ryanodine at a concentration (50 μM), which blocks the RyR. The second phase of contraction was attenuated by the anti-oxidant TEMPOL (3 mM), the NADPH oxidase inhibitor VAS2870 (10 μM), and the mitochondrial complex 3 blocker myxothiazole (1 μM). Incubation of cultured PA smooth muscle cells in 1 mM Na2S, followed by a wash to remove NaHS from the solution, led to a significant increase in cellular ROS production; this was not observed when expression of the mitochondrial membrane flavoprotein sulfide-quinone oxoreductase (SQR) was knocked down using siRNA. Low concentrations (10 and 30 μM) of Na2S hyperpolarized Ψ, whereas higher concentrations (0.1, 0.3, 0.5 and 1.0 mM) caused a triphasic response consisting of a brief hyperpolarization, followed by a depolarization and then a longer lasting hyperpolarization. The onset of the second contraction coincided with the second hyperpolarization, and also with the reversal of the sulfide-induced increase in NAD(P)H autofluorescence, indicative of recovery of the electron transport chain (ETC) from block. Based on these results, we conclude that both phase σ of sulfide-induced contraction involve PKC activation and release of Ca2+ from the sarcoplasmic reticulum via ryanodine receptors. The second phase of contraction also involves an increase in cellular ROS produced by complex 3, caused by an increased flow of electrons into the ETC consequent upon the metabolism of sulfide by SQR. http://dx.doi.org/10.1016/j.niox.2015.02.141
PP112 The role of hydrogen sulfide in the adherence of endothelial cells in vitro Panagiotis Panopoulos a, C. Malakis a, Andreas Papapetropoulos b, Stavros Topouzis a a Laboratory of Molecular Pharmacology, Department of Pharmacy, University of Patras, Greece
S59
b
Department of Pharmacy, National and Kapodistrian University of Athens, Athens, Greece
Hydrogen sulfide (H2S) is synthesized by a variety of mammalian tissues, by two pyridoxal 5′-phosphate-dependent enzymes that use L-cysteine as a substrate, namely cystathionine beta-synthase (CBS) and cystathionine gamma-lyase (CSE), and by the combined action of cysteine aminotransferase (CAT) and 3-mercaptopyruvate sulfurtransferase (3-MST). It has already been shown that in endothelial cells H2S can induce signaling responses which result in modulation of angiogenesis and changes in permeability. These effects require fine-tuning of key molecular interactions that determine the strength of cell–cell and cell–substrate adherence. In the current study we aimed to determine whether H2S affects the interaction of ECs to various substrates. To this goal, we first investigated the adherence or detachment of mouse brain endothelial cell (EC) line bEnd.3 to/from fibronectin (0.3 and 3 μg/ml) and gelatin (0.1 and 1 μg/ml) in the presence of 10 or 30 μM of NaHS, a treatment which results in physiological-like concentrations of released H2S. For adhesion assays, bEnd.3 cells were seeded in serum-free medium in the presence of the substrates and the H2S donor NaHS for 2 h, followed by washout and attached cell determination. For detachment assays, the cells were plated in complete media overnight and then were starved for 3 h in serum-free medium before treatment with the H2S donor NaHS for an additional 2 h. In both cases, the number of substrate-attached ECs was determined by incubation with the dye Crystal violet, extraction with 0.5% Triton-X and measurement of the OD at 630 nM. The amount of the extracted dye is directly proportional to cell number. Treatment with exogenous H2S did not significantly affect either the adherence or the detachment of bEnd.3 cells when either fibronectin or gelatin was used as substrate, in either concentrations of the H2S donor. Additional evidence on the effect of H2S on EC interactions with vitronectin and laminin will also be presented. Furthermore, parallel experiments evaluate the ability of endogenous H2S to modulate adhesion of bEnd.3 cells in all these substrates, by the use of aminooxyacetic acid (AOAA) and DL-propargylglycine (PAG) to inhibit the endothelial H2S-producing enzymes. Supported by a THALIS action: «Hydrogen sulphide, a new endogenous modulator of angiogenesis: investigating its signalling, (patho)physiological roles and development of novel pharmacological antagonists» Ö.Ê.:D554 / MIS: 380259, financed by EU and Greek national funds. http://dx.doi.org/10.1016/j.niox.2015.02.142
PP113 Physiological production of hydrogen sulfide is involved in porcine oocytes maturing in vitro Jan Nevoral a, Katerina Zamostna a, Tereza Zalmanova a, Veronika Kucerova-Chrpova a, Marketa Dvorakova a, Kristyna Hoskova a, Jaroslav Petr b a Czech University of Life Sciences in Prague, Department of Veterinary Sciences, Kamycka 129, 16521 Prague 6-Suchdol, Czech Republic b Research Institute of Animal Production, Pratelstvi 815, 10400 Prague 10-Uhrineves, Czech Republic Oocyte meiotic maturation is indispensable process of female gametogenesis. Hereby matured oocyte is predestined for fertilisation and subsequent embryonic development. The running of meiotic maturation is a result of activity of key factors – M-phase/Maturation Promoting Factor (MPF) and Mitogen Activated Protein Kinase (MAPK). Some of up-stream signal pathways, leading to key kinase activation, remain unsolved. Hydrogen sulfide signalling seems to
project P20664 and doctoral program BioToP W1224) and COST actions BM 1005 are greatly acknowledged. http://dx.doi.org/10.1016/j.niox.2015.02.140
PP111 Role of protein kinase C and reactive oxygen species in hydrogen sulfide mediated contraction of rat small pulmonary arteries Philip I. Aaronson, Jesus Prieto-Lloret, Yasin Shaifta, Vladimir Snetkov Division of Asthma Allergy & Lung Biology, King’s College London, London, UK The gas H2S (hereafter ‘sulfide’) acts as a vasodilator in the systemic circulation, but elicits a biphasic contraction in pulmonary arteries (PA) via an unknown mechanism. This may be a physiologically important effect; it has also been proposed that the sulfide concentration in PA increases during hypoxia, acting as an oxygen sensor which triggers hypoxic pulmonary vasoconstriction. We studied the mechanism of the sulfide-induced contraction in rings of rat 2nd order PA, using Na2S as a source of sulfide, by recording of isometric force development, the mitochondrial membrane potential (Ψ) using TMRE, and NAD(P)H autofluorescence, and also measured reactive oxygen species (ROS) levels in cultured PA smooth muscle cells using the luminescent ROS indicator LO12 (10 μM). Na2S (1 mM) applied in the presence of 5 μM PGF2a evoked a complex PA contraction comprising an initial transient contraction followed by a relaxation, and then a second and larger contraction which gradually relaxed. Both contractions persisted in the presence of the eNOS blocker L-NAME (300 μM), but were strongly antagonized by the protein kinase C blocker Gö6983 (3 μM) and by ryanodine at a concentration (50 μM), which blocks the RyR. The second phase of contraction was attenuated by the anti-oxidant TEMPOL (3 mM), the NADPH oxidase inhibitor VAS2870 (10 μM), and the mitochondrial complex 3 blocker myxothiazole (1 μM). Incubation of cultured PA smooth muscle cells in 1 mM Na2S, followed by a wash to remove NaHS from the solution, led to a significant increase in cellular ROS production; this was not observed when expression of the mitochondrial membrane flavoprotein sulfide-quinone oxoreductase (SQR) was knocked down using siRNA. Low concentrations (10 and 30 μM) of Na2S hyperpolarized Ψ, whereas higher concentrations (0.1, 0.3, 0.5 and 1.0 mM) caused a triphasic response consisting of a brief hyperpolarization, followed by a depolarization and then a longer lasting hyperpolarization. The onset of the second contraction coincided with the second hyperpolarization, and also with the reversal of the sulfide-induced increase in NAD(P)H autofluorescence, indicative of recovery of the electron transport chain (ETC) from block. Based on these results, we conclude that both phase σ of sulfide-induced contraction involve PKC activation and release of Ca2+ from the sarcoplasmic reticulum via ryanodine receptors. The second phase of contraction also involves an increase in cellular ROS produced by complex 3, caused by an increased flow of electrons into the ETC consequent upon the metabolism of sulfide by SQR. http://dx.doi.org/10.1016/j.niox.2015.02.141
PP112 The role of hydrogen sulfide in the adherence of endothelial cells in vitro Panagiotis Panopoulos a, C. Malakis a, Andreas Papapetropoulos b, Stavros Topouzis a a Laboratory of Molecular Pharmacology, Department of Pharmacy, University of Patras, Greece
S59
b
Department of Pharmacy, National and Kapodistrian University of Athens, Athens, Greece
Hydrogen sulfide (H2S) is synthesized by a variety of mammalian tissues, by two pyridoxal 5′-phosphate-dependent enzymes that use L-cysteine as a substrate, namely cystathionine beta-synthase (CBS) and cystathionine gamma-lyase (CSE), and by the combined action of cysteine aminotransferase (CAT) and 3-mercaptopyruvate sulfurtransferase (3-MST). It has already been shown that in endothelial cells H2S can induce signaling responses which result in modulation of angiogenesis and changes in permeability. These effects require fine-tuning of key molecular interactions that determine the strength of cell–cell and cell–substrate adherence. In the current study we aimed to determine whether H2S affects the interaction of ECs to various substrates. To this goal, we first investigated the adherence or detachment of mouse brain endothelial cell (EC) line bEnd.3 to/from fibronectin (0.3 and 3 μg/ml) and gelatin (0.1 and 1 μg/ml) in the presence of 10 or 30 μM of NaHS, a treatment which results in physiological-like concentrations of released H2S. For adhesion assays, bEnd.3 cells were seeded in serum-free medium in the presence of the substrates and the H2S donor NaHS for 2 h, followed by washout and attached cell determination. For detachment assays, the cells were plated in complete media overnight and then were starved for 3 h in serum-free medium before treatment with the H2S donor NaHS for an additional 2 h. In both cases, the number of substrate-attached ECs was determined by incubation with the dye Crystal violet, extraction with 0.5% Triton-X and measurement of the OD at 630 nM. The amount of the extracted dye is directly proportional to cell number. Treatment with exogenous H2S did not significantly affect either the adherence or the detachment of bEnd.3 cells when either fibronectin or gelatin was used as substrate, in either concentrations of the H2S donor. Additional evidence on the effect of H2S on EC interactions with vitronectin and laminin will also be presented. Furthermore, parallel experiments evaluate the ability of endogenous H2S to modulate adhesion of bEnd.3 cells in all these substrates, by the use of aminooxyacetic acid (AOAA) and DL-propargylglycine (PAG) to inhibit the endothelial H2S-producing enzymes. Supported by a THALIS action: «Hydrogen sulphide, a new endogenous modulator of angiogenesis: investigating its signalling, (patho)physiological roles and development of novel pharmacological antagonists» Ö.Ê.:D554 / MIS: 380259, financed by EU and Greek national funds. http://dx.doi.org/10.1016/j.niox.2015.02.142
PP113 Physiological production of hydrogen sulfide is involved in porcine oocytes maturing in vitro Jan Nevoral a, Katerina Zamostna a, Tereza Zalmanova a, Veronika Kucerova-Chrpova a, Marketa Dvorakova a, Kristyna Hoskova a, Jaroslav Petr b a Czech University of Life Sciences in Prague, Department of Veterinary Sciences, Kamycka 129, 16521 Prague 6-Suchdol, Czech Republic b Research Institute of Animal Production, Pratelstvi 815, 10400 Prague 10-Uhrineves, Czech Republic Oocyte meiotic maturation is indispensable process of female gametogenesis. Hereby matured oocyte is predestined for fertilisation and subsequent embryonic development. The running of meiotic maturation is a result of activity of key factors – M-phase/Maturation Promoting Factor (MPF) and Mitogen Activated Protein Kinase (MAPK). Some of up-stream signal pathways, leading to key kinase activation, remain unsolved. Hydrogen sulfide signalling seems to