P11 Hydrogen sulfide and its effect on the hypoxic response in porcine coronary arteries

Abstract

Hydrogen sulfide (H2S) is synthesised via 3 main enzymes: cystathionine γ-lyase (CSE), cystathionine β-synthase (CBS) and 3-mercaptopyruvate sulfurtransferase (3-MST) and has been shown to act as an oxygen sensor/transducer in the vasculature [1] . Our preliminary studies show a role for H2S in mediating porcine coronary artery vasodilation induced by hypoxia, mainly through production via CBS [2] . The aim of the current study was to expand these findings to investigate whether 3-MST is involved in the hypoxic response and determine expression and location of all three H2S-producing enzymes in the porcine coronary artery. Segments of porcine coronary arteries, obtained from a local abattoir, were mounted in organ baths, containing Krebs-Henseleit buffer (37°C) gassed with 95% O2/5% CO2, for isometric tension recording. Viability was assessed using 60 mM KCl. Arteries were precontracted with the thromboxaneA2 mimetic, U46619; once a stable tone (∼50% of KCl response) was achieved, hypoxia was induced using 95% N2/5% CO2 for 30 min. Hypoxic responses were investigated in the absence and presence of inhibitors of CSE, CBS and/or 3-MST. Western blotting and immunohistochemistry were used to assess expression and localisation of CSE, CBS and 3-MST. Data were presented as mean ± SEM with contractions expressed as a percentage of the response to KCl. Relaxations were expressed as a percentage of the U46619-induced tone or as area under the curve (AUC). Data were analysed using Student’s t test where P Exposing coronary arteries to hypoxia caused a biphasic response; a transient contraction followed by a prolonged relaxation. The CSE inhibitor D,L-propargylgylcine (PPG 10 μM) and the competitive MPST inhibitor aspartate (1 mM) alone or in combination had no effect on the vascular response to hypoxia. The CBS inhibitor amino-oxyacetate (AOAA) alone (100 μM) caused a significant reduction in the contractile phase of the hypoxic response (control 14 ± 2%, +AOAA 10 ± 3%, P = 0.02). However, this residual response to AOAA was further reduced in the presence of PPG, aspartate or both (Contraction: control 12 ± 2% vs. PPG/AOAA 6 ± 2% P = 0.02, control 14 ± 2% vs. PPG/AOAA/aspartate 7 ± 2% P = 0.01. Relaxation: control 62 ± 5% vs. PPG/AOAA 46 ± 6% P = 0.02, control 76 ± 6% vs. AOAA/aspartate 46 ± 6% P = 0.001 and control 60 ± 5% vs. PPG/AOAA/aspartate 42 ± 5% P = 0.006). All three enzymes were expressed in the coronary artery. These data show that H2S is an endogenous mediator of the hypoxic response in porcine coronary arteries. These data also show that all three H2S-producing enzymes are present within the coronary artery and suggest that the most important enzyme responsible for H2S generation during the hypoxic response in the coronary artery is CBS. However, there is some contribution from other H2S-producing enzymes, CSE and MPST, but this only becomes apparent when coupled with CBS blockade.