No role for hydrogen sulphide in the anticontractile effect of perivascular adipose tissue in porcine coronary artery.

A. A. Ahmad, M. D. Randall, R. E. Roberts.School of Life Sciences, University of Nottingham, Nottingham, United Kingdom.

Introduction: Hydrogen sulphide (H₂S) has been described as a novel candidate for an adipose derived relaxing factor (ADRF) in rats and mice¹. However, there are controversial findings in the expression and function of H₂S synthesizing enzymes in different species². We have previously demonstrated that perivascular adipose tissue (PVAT) inhibits thromboxane receptor-mediated contractions in the porcine coronary artery (PCA)³. Therefore, the aim of this study was to determine the role of H₂S in PVAT-induced vasorelaxation of the porcine coronary arterial (PCA) tone.

Method: Western immunoblotting was performed to investigate the expression of the different H₂S synthesizing enzymes (CSE, CBS and 3-MPST) in PVAT. Contractile responses to the thromboxane mimetic U46619 in segments of porcine coronary artery with and without PVAT were recorded in an isometric tension recording system. H₂S synthesizing enzymes activity in PVAT was estimated using SF7-AM fluorescence assay. A Student’s 2-tailed paired t-test was used to analyse the data. Data are expressed as mean±S.E.M.

Results: Western immunoblotting showed that CBS and 3-MPST are expressed in PVAT whilst no CSE expression was identified (n=8). Contractions to the thromboxane mimetic U46619 were reduced in the presence of PVAT (Rmax = 82± 5% with PVAT vs Rmax = 106± 8% of vessels lacking PVAT (P<0.01)). Besides, log EC50% was also significantly reduced in the rings with fat attached (pEC50 = 7.9 ± 0.06) compared to control (pEC50 =8.2± 0.04) (P<0.005) (n=12). Pre-incubation with the hydrogen sulphide enzyme inhibitors 4-propargylglycine (PPG) (10μM) and 2-(aminooxy)-acetic acid (AOAA) (100μM) did not prevent this anticontractile response to PVAT (n=6). Assessment of H₂S synthesis using the fluorescent tag SF7 demonstrated CBS activity in PVAT, which was inhibited by AOAA (5.6±0.15 fluorescence units in control vs 5.2±0.1 with AOAA, n=5). 3-MPST activity was also detected in PVAT (61.6±5.7 fluorescence units, n=5). However, the activities of these enzymes in PVAT were much lower in comparison with the activities of the same enzymes in rat liver (CBS, 26.5 control vs 5.0 with AOAA; 3-MPST, 138.6).

Conclusion: H₂S can be synthesised in PVAT in the PCA through CBS and 3-MPST, but has no role in the anti-contractile effect of PVAT.

References:

1. Fang et al. (2009). J Hypertens 27: 2174-85.

2. Kohn et al. (2012). PLoS One 7: e41951.

3. Ahmad et al. (2017). Br J Pharmacol 16: 2773-2783.