H2S as a physiologic vasorelaxant: hypertension in mice with deletion of cystathionine gamma-lyase

Abstract

Studies of nitric oxide over the past two decades have highlighted the fundamental importance of gaseous signaling molecules in biology and medicine. The physiological role of other gases such as carbon monoxide and hydrogen sulfide (H2S) is now receiving increasing attention. Here we show that H2S is physiologically generated by cystathionine gamma-lyase (CSE) and that genetic deletion of this enzyme in mice markedly reduces H2S levels in the serum, heart, aorta, and other tissues. Mutant mice lacking CSE display pronounced hypertension and diminished endothelium-dependent vasorelaxation. CSE is physiologically activated by calcium-calmodulin, which is a mechanism for H2S formation in response to vascular activation. These findings provide direct evidence that H2S is a physiologic vasodilator and regulator of blood pressure.

Fig. 1

Phenotype of CSE male knockout mice. (A) Reduced H₂S production from aorta and heart tissues in CSE^−/− mice and CSE^−/+ mice. Number of mice are given for each group; n = 16. (B) Reduced serum H₂S level in CSE^−/− mice and CSE^−/+ mice (n = 8 to 10). (C) Age-dependent increase in blood pressure of CSE^−/− mice and CSE^−/+ mice (n = 12). (D) H₂S administration lowers systolic arterial blood pressure in 10-week-old CSE^−/− mice (n = 13 to 15). (E) Increased plasma homocysteine level in CSE^−/− mice and CSE^−/+ mice (n = 19). (F) Decreased plasma l-cysteine level in CSE^−/− mice and CSE^−/+ mice (n = 15). All results are means ± SEM. *P < 0.05 versus WT; #P < 0.05 versus heterozygote.

Fig. 2

Impaired endothelial function in CSE mutant mice. Contraction of mesenteric artery evoked by phenylephrine (A) and relaxation of mesenteric artery by sodium nitroprusside (B), H₂S (C), and methacholine (D). n = 15 for each group. All results are means ± SEM. *P < 0.05 versus WT; #P < 0.05 versus heterozygote. (E) Endothelial removal abolishes methacholine-induced relaxation of mesenteric artery. No relaxation occurs in vessels of WT or mutant mice after stripping of the endothelium. For CSE^−/− mice, n = 8; and for CSE^+/+ mice, n = 9. (F) Immunohistochemical localization of CSE to arterial endothelium (black arrows) is abolished in CSE^−/− mice. Scale bars, 20 µm.

Fig. 3

CSE is activated by calcium-calmodulin upon muscarinic cholinergic stimulation of vascular endothelial cells. (A) Calmodulin binds CSE in vitro in the presence of calcium (2 mM). The interaction is diminished by the calcium chelator, EGTA (1 mM), as well as the calmodulin antagonist W7 (100 µM). (B) Calcium-calmodulin activates purified CSE in vitro. Calcium (1 mM) or calmodulin (5 µM) separately has no effect on CSE activity. n = 3. *P < 0.05 versus the control. (C) CSE is endogenously expressed in bovine aortic endothelial cells (BAECs) and human umbilical vein endothelial cells (HUVECs). (D) CSE is activated in BAECs treated with the calcium ionophore A23187 (1 µM) for 10 min. Incubation beforehand with the acetoxy-methylester of the intracellular calcium chelator BAPTA (BAPTA-AM. 50 µM) or W7 (50µmM) for 30 min prevents CSE activation. n = 3. *P < 0.05 versus A23187 treatment; #P < 0.05 versus control. (E) CSE is strongly activated in BAECs treated with the muscarinic agonist methacholine (MCh, 1 µM) for 10 min. The activation is twice as much as with similar concentrations of A23187. The stimulatory effect of MCh is abolished by the muscarinic antagonist atropine (50 µM) (n = 3 or 4). *P < 0.05 versus control; #P < 0.05 versus all other groups. (F) CSE is the endogenousH₂S generator in BAECs. Transfecting cells with 100 nM CSE-specific short interfering RNA (CSE-siRNA) for 48 hours markedly diminishes the enhanced H₂S production observed with A23187 (1 µM) or MCh (1 µM). Western blotting showed that CSE protein is decreased about 60 to 70% by CSE-siRNA (n = 3). *P < 0.05.