Hydrogen sulfide-mediated endothelial function and the interaction with eNOS and PDE5A activity in human internal mammary arteries

Figure 1.. (A) Effect of cysteine aminotransferase (CAT) inhibitors on endogenous H2S-induced relaxation. Acetylcholine (ACh) induced almost full relaxation in human endothelium-intact (E+) internal mammary artery (IMA) rings, and this relaxation was inhibited by NG-nitro-L-arginine (L-NNA) plus indomethacin (Indo) (**P <0.01, two-way ANOVA); ACh-induced relaxation in E+ rings was further inhibited by L-NNA plus Indo plus aminooxyacetic acid (AOAA; a general inhibitor of pyridoxal 5′-phosphate-dependent enzymes), and L-NNA plus Indo plus L-aspartate (a competitive CAT substrate). In contrast, there was no ACh-induced relaxation in endothelium-denuded (E-) IMA rings (n = 8; **P <0.01 versus E+; two-way ANOVA); (B) Original trace of Figure 1A; (C) –Log EC50 for each group; L-NNA plus Indo plus AOAA was significantly lower than L-NNA plus Indo (**P <0.01; n = 8; one-way ANOVA and Bonferroni test). Data presented as mean ± SEM.

Figure 2.. (A) Standard curve of NaHS gradient solutions that was produced to quantify the concentration of H2S in fresh human internal mammary artery (IMA) tissue; (B) The concentrations of H2S in human IMA were increased by incubating with acetylcholine (ACh) for 4 h (**P <0.01 versus control; n = 5; unpaired t-test). Data presented as mean ± SEM.

Figure 3.. Representative photomicrographs showing immunohistochemistry staining of 3-mercaptopyruvate sulfurtransferase (3-MPST) and cysteine aminotransferase (CAT) in human internal mammary artery tissue; 3-MPST was shown to be located in both the endothelium and smooth muscle (left); cytosolic CAT (cCAT) was shown to be located only in the endothelial cells (middle) and mitochondrial CAT (mCAT) was found to be located in both the endothelium and smooth muscle (right); original magnification × 200.

Figure 4.. Effect of different K+ channel blockers on acetylcholine (ACh)-induced relaxation in human internal mammary artery rings. (A) ACh-induced relaxation was significantly reduced in the presence of NG-nitro-L-arginine (L-NNA) plus indomethacin (Indo). The K+ channel blockers iberiotoxin, glibenclamide and TRAM-34 plus apamin further inhibited the residual ACh-induced relaxation that was resistant to L-NNA plus Indo (**P <0.01 versus L-NNA plus Indo [control]; two-way ANOVA); (B) Regarding –logEC50 values, each experimental group had significantly higher EC50 values versus the control group (L-NNA plus Indo) except L-NNA plus Indo plus iberiotoxin, in which the EC50 was lower (**P <0.01 versus control; n = 8; one-way ANOVA and Bonferroni test). Data presented as mean ± SEM.

Figure 5.. The NaHS-mediated relaxation in human internal mammary artery (IMA) rings was not affected by denudation of the endothelium (P >0.05, endothelium-intact IMA versus endothelium-denuded IMA; n = 8; two-way ANOVA). Data presented as mean ± SEM.

Figure 6.. Levels of endothelial nitric oxide synthase (eNOS), phosphorylated (p)-eNOS, and phosphodiesterase (PDE)5A in human internal mammary artery (IMA) rings. (A) Top panel shows representative Western blots of IMA protein extracts from IMA rings that had been incubated with Krebs alone (control; two left lanes) or Krebs with −4 log M NaHS (two right lanes) for 24 h. Results normalised to GAPDH (bottom figure) showed that exogenous H2S did not affect eNOS levels but increased p-eNOSser1177 levels in human IMA (**P <0.01 versus control; n = 5; unpaired t-test). (B) Results from enzyme-linked immunosorbent assay showing that stimulation of IMA with NaHS (−4 log M) significantly down-regulated PDE5A levels compared with the control (**P <0.01 versus control; n = 5; unpaired t-test). Data presented as mean ± SEM.