br Introduction Hydrogen sulfide H S
Introduction Hydrogen sulfide (H2S) and nitric oxide (NO) are important gasotransmitters in the cardiovascular system and instrumental to the fine control of vascular tone  and cellular function . NO is synthesized from l-arginine by calmodulin-dependent endothelial nitric oxide synthase (eNOS) in the vascular endothelium . H2S is generated within the mammalian cells mainly via enzymatic pathways, although non-enzymatic production (by reduction of thiols and thiol-containing molecules) is also possible , . H2S producing enzymes include cystathionine β-synthase (CBS), cystathionine γ-lyase (CSE) and 3-mercaptopyruvate sulfur transferase/cysteine aminotransferase (MST/CAT) and are known to be expressed throughout the body , , , . CSE is the dominant H2S generating enzyme in vasculature and is expressed in the endothelium as well as vascular smooth muscle cells . The relaxant role of H2S has been demonstrated in several vessels but the mechanism of action is unclear with the roles being attributed to KATP channels, Ca2+ channels and increased vascular cGMP levels as a consequence of PDE inhibition , , , , , . There is now growing evidence that H2S can alter the biological response of NO in a tissue specific way, as H2S stimulates eNOS activity in mouse 516 3 and endothelial cells , , ,  but not in rabbit heart , . Although the influence of H2S on the regulation of NO formation has been studied in detail , , , , , , few studies have focused on the consequences of NO deficiency or its increased generation on H2S-induced relaxations. Pharmacologic inhibition or genetic deletion of eNOS has been reported to decrease relaxations to the exogenous H2S donor NaHS in rat aorta , . Nevertheless, there are also contradictory studies showing increased expression of CSE and CBS and relaxation to NaHS in non-obese diabetic rat aorta  or enhanced relaxation to l-cysteine in diabetic cerebral arteries, where severe endothelial dysfunction is observed in both cases . Moreover, we recently reported that, the l-cysteine-induced formation of H2S and subsequent relaxation as well as the expression of CSE and MPST were increased in penile tissues from eNOS−/− mice . However, the effects of H2S on the regulation of vascular tone are tissue specific as shown by increased H2S formation and related enzyme expression in response to estrogen in sheep uterine and mesenteric artery but lack of this effect in carotid arteries from the same animals . Thus in the present study, we investigated the consequences of altered eNOS expression on endogenous or exogenous H2S-induced regulation of vascular tonus in mice carotid arteries using eNOS deficient species, adenoviral transfection technique and a pharmacological inhibitor of H2S producing enzymes.
Materials and methods
Discussion The main finding in our study is that deletion of eNOS increased but overexpression/replacement of eNOS decreased l-cysteine-induced relaxation in mice carotid arteries. There was a compensatory balance between l-cysteine-induced and exogenous H2S donor-induced relaxations in a way that a decrease in relaxation to l-cysteine compensated by enhanced relaxation to exogenous H2S and vice versa. l-cysteine is a substrate of H2S enzymes but has other physiological roles. l-cysteine relaxed murine carotid arteries at a concentration of 1 mM in eNOS−/− and 3 mM in the control group (Fig. 3). Its concentration in human or mouse plasma is also reported to be in the range of 0.23–3.3 mM , , , . Thus the concentrations required to elicit l-cysteine-induced relaxation in murine carotid arteries were similar to those reported in plasma. l-cysteine-induced H2S formation is well described and has been reported in other murine vessels , ,  as well as other species , , , ,  As far as the authors are aware there is no study directly demonstrating H2S formation in murine carotid arteries, probably due to difficulties with material amounts. However l-cysteine induced H2S-dependent relaxation and/or H2S formation has been demonstrated in carotid arteries from rats ,  and sheep . Besides inhibition of l-cysteine-induced relaxations and H2S formation by H2S inhibitors are in the same rate , suggesting the relaxation induced by l-cysteine is H2S dependent.