cGMP-dependent protein kinase contributes to hydrogen sulfide-stimulated vasorelaxation

A Cantalupo1, E Mitidieri1, V Vellecco1, MR Bucci1, A Papapetropoulos2, R Wang3, R Feil4, G Cirino1. 1University of Naples Federico II, Experimental Pharmacology 80131, Italy, 2University of Patras, Department of Pharmacy, Greece, 3Lakehead University, Department of Biology, Canada, 4Universität Tübingen, Interfakultäres Institut für Biochemie, Germany

Background and aim. A growing body of evidence suggests that hydrogen sulfide (H2S) is a signaling molecule in mammalian cells. In the cardiovascular system, H2S enhances vasodilatation and angiogenesis (Szabo and Papapetropoulos, 2011). H2S-induced vasodilatation is hypothesized to occur through ATP-sensitive potassium channels; however, recently it has been demonstrated that H2S also increases cGMP levels in tissues (Bucci et al., 2010). Herein, we studied the involvement of cGMPdependent protein kinase-I in H2S-induced vasorelaxation.

Methods and results. Pretreatment of aortic rings with sildenafil attenuated NaHS-induced relaxation (Emax 75.2 ± 12.2% vs. 91.3 ± 7.2%, sildenafil and vehicle respectively; n=4), confirming previous findings that H2S is a phosphodiesterase inhibitor. In line with the notion that H2S is an inhibitor of phosphodiesterase, vascular tissue levels of cGMP in cystathionine gamma lyase (CSE) knockouts were lower than those in wild-type control mice (aorta 9.55 ± 0.80 pmole/mg vs. 23.40 ± 1.44 pmole/mg protein, CSE-/- mice vs. wild-type mice respectively; n=5; mesenteric artery 0.42 ± 0.04 pmole/mg protein vs. 1.57 ± 0.05 pmole/mg protein, CSE-/- mice vs. wild-type mice respectively; n=5). Treatment of aortic rings with NaHS, a fast releasing H2S donor, enhanced phosphorylation of vasodilator-stimulated phosphoprotein in a time-dependent manner, suggesting that cGMP-dependent protein kinase (PKG) is activated after exposure to H2S. Incubation of rings with a PKG-I inhibitor (DT-2) attenuated NaHS-stimulated relaxation (Ec50 1.9x10-4M vs. 7.1x10-5M DT-2 vs vehicle respectively; n=6). Interestingly, vasodilatory responses to a slowly releasing H2S donor (GYY 4137) were unaffected by DT-2 (46.6 ± 8.8 vs. 32.2 ± 9.4 vehicle and DT-2 respectively, n=7), suggesting that this donor dilates mouse aorta through PKG-independent pathways. In agreement to what was observed with DT-2, dilatory responses to NaHS and L-cysteine (a substrate for H2S production) were reduced in vessels of PKG-I knockout mice (NaHS, 92.0 ± 3.0 vs. 68.3 ± 3.6 WT and PKG-KO respectively, n=7; L-cys, 23.1 ± 2.1 vs. 10.0 ± 2.8 WT and PKG-KO respectively, n=12).

Conclusion. Our results confirm the role of cGMP in the vascular responses to H2S and demonstrate that PKG-I regulates H2S-stimulated vasodilation.