BACKGROUND AND PURPOSETo investigate whether diabetes affects either or both nitric oxide (NO)-mediated and endothelium-derived hyperpolarizing factor (EDHF)-type relaxation in endothelium-dependent relaxation of mesenteric arteries from streptozotocin-induced diabetic rats. EXPERIMENTAL APPROACHWire myography was employed to examine endothelial function of mesenteric arteries. Superoxide levels were measured by L-012 and lucigenin-enhanced chemiluminescence. Western blotting was used to quantify protein expression levels. KEY RESULTSSuperoxide levels were significantly increased in diabetic mesenteric arteries compared with normal arteries. Diabetes significantly reduced the sensitivity to the endothelium-dependent relaxant, acetylcholine (ACh) in mesenteric arteries. When the contribution of NO to relaxation was abolished by N-nitro-L-arginine (L-NNA) + a soluble guanylate cyclase inhibitor (ODQ), the sensitivity to ACh was significantly decreased in the diabetic arteries compared with normal arteries, indicating an impaired EDHF-type relaxation despite increased expression of intermediate-and small-conductance calcium-activated potassium channels. Conversely, when the contribution of EDHF was inhibited with TRAM-34 + apamin + iberiotoxin, maximum relaxations to ACh were significantly decreased in diabetic compared with normal arteries, suggesting that the contribution of NO was also impaired by diabetes. Basal levels of NO release, indicated by contraction to L-NNA, were also significantly decreased in diabetic arteries. Western blot analysis demonstrated that diabetic arteries had an increased expression of Nox2, decreased pSer 473 Akt and a reduced proportion of endothelial NO synthase (eNOS) expressed as a dimer, indicating uncoupling. CONCLUSION AND IMPLICATIONSThe contribution of both NO and EDHF-type relaxations was impaired in diabetes and was caused by increased oxidative stress, decreased pSer 473 Akt and/or eNOS uncoupling. AbbreviationsEDHF, endothelium-derived hyperpolarizing factor; eNOS, endothelial nitric oxide

Hydrogen sulfide (H2S) is increasingly recognized as a gasotransmitter with protective effects in the cardiovascular system. The aim of the study was to examine the effects of chronic NaHS treatment on blood pressure, vascular function and oxidative stress in an in vivo model of hypertension and oxidative stress. Male C57Bl6/J mice were rendered hypertensive with 0.7 mg kg(-1) per day angiotensin II (AngII) for 14 days administered via implanted mini-pumps. The mice were treated with NaHS (10 μmol kg(-1) per day) to deliver H2S or an inhibitor of cystathionine-γ-lyase, DL-propargylglycine (PPG 30 mg kg(-1) per day) via intraperitoneal (i.p.) injection. Systolic blood pressure was measured and vascular function examined by myography. Vascular superoxide production was measured by lucigenin-enhanced chemiluminescence. AngII infusion significantly increased systolic blood pressure (P < 0.001). This increase was significantly attenuated by treatment with NaHS (P < 0.001). Both aortic endothelial function and NO bioavailability were significantly attenuated in the AngII group (P < 0.01) but this attenuation was reversed by NaHS treatment. Similarly, aortic superoxide anion production was significantly enhanced by AngII (P < 0.01), and this was reversed by NaHS treatment, and also exacerbated by PPG treatment (P < 0.001). These data show that in a mouse model of hypertension and oxidative stress induced by AngII, exogenous H2S treatment in vivo reduces blood pressure, endothelial dysfunction and vascular oxidative stress, while inhibiting endogenous H2S production in vivo is deleterious. This furthers the evidence that H2S is a vasoprotective molecule that may be a useful treatment target in cardiovascular disease.

This study aimed to elucidate the molecular mechanism of H(2)S-induced vasorelaxation. Vasorelaxation responses to the H(2)S donor NaHS and the H(2)S precursor L: -cysteine were examined by measuring isometric tone of mouse aortic rings in a small vessel myograph. H(2)S concentrations in Krebs' solution were determined with a polarographic sensor. H(2)S expression was examined by Western blot, and H(2)S production from CSE was assayed using a spectroscopic method. In pre-constricted mouse aorta, NaHS (1 μM-3 mM) elicited vasorelaxation of 95 ± 7%, EC(50) 189 ± 69 μM. This response was unaffected by removal of the endothelium. Maximum vasorelaxation was significantly attenuated by global blockade of K(+) channels (50 mM K(+)) and the K(ATP) channel blocker glibenclamide (10 μM) alone (P < 0.01, ANOVA). Specific inhibition of K(Ca), K(IR), or K(V) channels elicited a significant shift to the right in the concentration-response curve to NaHS (P < 0.01, ANOVA) without affecting maximum relaxation. NaHS-mediated vasorelaxation was inhibited by the Cl(-) channel inhibitor DIDS (1 mM, P < 0.05, t test), and NaHS caused a significant concentration-dependent inhibition of voltage-gated Ca(2+) channels (P < 0.001, two-way ANOVA). The H(2)S-producing enzyme cystathionine-γ-lyase (CSE) was expressed in mouse aorta and had activity of 7 ± 3 μmol H(2)S/g/min. L: -cysteine (1 μM-3 mM) elicited a CSE-dependent vasorelaxation of mouse aorta with intact endothelium (20 ± 7%), but not when the endothelium was removed. CSE inhibitors DL: -propargylglycine (20 mM) and β-cyanoalanine (1 mM) caused concentration-dependent contraction of mouse aorta. In mouse aorta, H(2)S elicits endothelium-independent vasorelaxation involving several different ion channels and seems to converge at the vascular smooth muscle cell voltage-gated Ca(2+) channel. The L: -cysteine-CSE-H(2)S pathway contributes to vasorelaxation and appears to modulate basal vessel tone.

Background3',4'-Dihydroxyflavonol (DiOHF) is an effective antioxidant that acutely preserves nitric oxide (NO) activity in the presence of elevated reactive oxygen species (ROS). We hypothesized that DiOHF treatment (7 days, 1 mg/kg per day s.c.) would improve relaxation in mesenteric arteries from diabetic rats where endothelial dysfunction is associated with elevated oxidant stress.Methodology/Principal FindingsIn mesenteric arteries from diabetic rats there was an increase in ROS, measured by L-012 and 2',7'-dichlorodihydrofluorescein diacetate fluorescence. NADPH oxidase-derived superoxide levels, assayed by lucigenin chemiluminescence, were also significantly increased in diabetic mesenteric arteries (diabetes, 4892±946 counts/mg versus normal 2486±344 counts/mg, n = 7–10, p<0.01) associated with an increase in Nox2 expression but DiOHF (2094±300 counts/mg, n = 10, p<0.001) reversed that effect. Acetylcholine (ACh)-induced relaxation of mesenteric arteries was assessed using wire myography (pEC50 = 7.94±0.13 n = 12). Diabetes significantly reduced the sensitivity to ACh and treatment with DiOHF prevented endothelial dysfunction (pEC50, diabetic 6.86±0.12 versus diabetic+DiOHF, 7.49±0.13, n = 11, p<0.01). The contribution of NO versus endothelium-derived hyperpolarizing factor (EDHF) to ACh-induced relaxation was assessed by evaluating responses in the presence of TRAM-34+apamin+iberiotoxin or N-nitro-L-arginine+ODQ respectively. Diabetes impaired the contribution of both NO (maximum relaxation, Rmax diabetic 24±7 versus normal, 68±10, n = 9–10, p<0.01) and EDHF (pEC50, diabetic 6.63±0.15 versus normal, 7.14±0.12, n = 10–11, p<0.01) to endothelium-dependent relaxation. DiOHF treatment did not significantly affect the EDHF contribution but enhanced NO-mediated relaxation (Rmax 69±6, n = 11, p<0.01). Western blotting demonstrated that diabetes also decreased expression and increased uncoupling of endothelial NO synthase (eNOS). Treatment of the diabetic rats with DiOHF significantly reduced vascular ROS and restored NO-mediated endothelium-dependent relaxation. Treatment of the diabetic rats with DiOHF also increased eNOS expression, both in total and as a dimer.Conclusions/SignificanceDiOHF improves NO activity in diabetes by reducing Nox2-dependent superoxide production and preventing eNOS uncoupling to improve endothelial function.

Hydrogen sulfide (H(2)S) is an endogenous mediator with peripheral vasorelaxant effects; however, the mechanism of H(2)S-induced vasorelaxation in cerebral blood vessels has not been extensively studied. Vasorelaxation studies were performed on middle cerebral arteries from male Sprague Dawley rats using wire myography. Immunofluorescence staining was used to detect the presence of the H(2)S-producing enzyme cystathionine-γ-lyase (CSE). CSE was present in the endothelium and smooth muscle of middle cerebral arteries. The CSE substrate, L-cysteine, induced vasorelaxation that was sensitive to the CSE inhibitor DL-propargylglycine. This relaxation was independent of endothelium, suggesting that H(2)S was produced in the vascular smooth muscle. The H(2)S donor, sodium hydrogen sulfide (NaHS; 0.1-3.0 mM) produced concentration-dependent relaxation, which was unaffected by endothelium removal. Nifedipine (3 μM) significantly reduced the maximum relaxation elicited by NaHS. Inhibiting potassium (K(+)) conductance with 50 mM K(+) significantly attenuated NaHS-induced relaxation, however, selective blockers of ATP sensitive (K(ATP)), calcium sensitive (K(Ca)), voltage dependent (K(V)), or inward rectifier (K(ir)) channels alone or in combination did not affect the response to NaHS. 4,4-diisothiocyanatostilbene-2,2-disulfonic acid (DIDS; 300 μM) caused a significant rightward shift of the NaHS concentration-response curve, but this effect could not be explained by inhibition of Cl(-) channels or Cl(-)/HCO (3)(-) exchange, as selective blockade of these mechanisms had no effect. These findings suggest endogenous H(2)S can regulate cerebral vascular function. The H(2)S-mediated relaxation of middle cerebral arteries is DIDS sensitive and partly mediated by inhibition of L-type calcium channels, with an additional contribution by K channels but not K(ATP), K(Ca), K(V), or K(ir) subtypes.

The aim of this study was to examine the ability of H2S, released from NaHS to protect vascular endothelial function under conditions of acute oxidative stress by scavenging superoxide anions (O2(-)) and suppressing vascular superoxide anion production. O2(-) was generated in Krebs' solution by reacting hypoxanthine with xanthine oxidase (Hx-XO) or with the O2(-) generator pyrogallol to model acute oxidative stress in vitro. O2(-) generation was measured by lucigenin-enhanced chemiluminescence. Functional responses in mouse aortic rings were assessed using a small vessel myograph. NaHS scavenged O2(-) in a concentration-dependent manner. Isolated aortic rings exposed to either Hx-XO or pyrogallol displayed significantly attenuated maximum vasorelaxation responses to the endothelium-dependent vasodilator acetylcholine, and significantly reduced NO bioavailability, which was completely reversed if vessels were pre-incubated with NaHS (100 μM). NADPH-stimulated aortic O2(-) production was significantly attenuated by the NADPH oxidase inhibitor diphenyl iodonium. Prior treatment of vessels with NaHS (100 nM-100 μM; 30 min) inhibited NADPH-stimulated aortic O2(-) production in a concentration-dependent manner. This effect persisted when NaHS was washed out prior to measuring NADPH-stimulated O2(-) production. These data show for the first time that NaHS directly scavenges O2(-) and suppresses vascular NADPH oxidase-derived O2(-) production in vitro. Furthermore, these properties protect endothelial function and NO bioavailability in an in vitro model of acute oxidative stress. These results suggest that H2S can elicit vasoprotection by both scavenging O2(-) and by reducing vascular NADPH oxidase-derived O2(-) production.

Gaseous mediators are important signaling molecules with properties that differ from other, larger signaling molecules. Small gaseous mediators readily cross cell membranes and can access sites on target molecules that would be inaccessible to bulkier molecules. They have a variety of signaling mechanisms, some well understood, some not. The family of gasotransmitters is growing, well known members include nitric oxide (NO) and carbon monoxide (CO). Newer candidates include the sulfur containing gases hydrogen sulfide (H2S), which has been shown to have a wide range of physiological functions, and more recently sulfur dioxide (SO2) has been studied as a potential new gasotransmitter. This review explores the production, regulation and role of the sulfur-containing gases H2S and SO2 at the level of the endothelial and vascular smooth muscle cells as well as the broader effects on the cardiovascular system under both physiological and pathophysiological conditions.

Hydrogen sulfide is a novel mediator with the unique properties of a gasotransmitter and many and varied physiological effects. Included in these effects are a number of cardiovascular effects that are proving beneficial to vascular health. Specifically, H2S can elicit vasorelaxation, prevention of inflammation and leukocyte adhesion, anti-proliferative effects and anti-thrombotic effects. Additionally, H2S is a chemical reductant and nucleophile that is capable of inhibiting the production of reactive oxygen species, scavenging and neutralising reactive oxygen species and boosting the efficacy of endogenous anti-oxidant molecules. These result in resistance to oxidative stress, protection of vascular endothelial function and maintenance of blood flow and organ perfusion. H2S has been shown to be protective in hypertension, atherosclerosis and under conditions of vascular oxidative stress, and deficiency of endogenous H2S production is linked to cardiovascular disease states. Taken together, these effects suggest that H2S has a physiological role as a vasculoprotective factor and that exogenous H2S donors may be useful therapeutic agents. This review article will discuss the vascular effects and anti-oxidant properties of H2S as well as examine the protective role of H2S in some important vascular disease states.