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  • We should also note that


    We should also note that, although AOAA is not a specific H2S inhibitor, its inhibitory effect on l-cysteine-induced relaxations are related to H2S since addition of AOAA to another H2S inhibitor PAG did not provide a further inhibition of these relaxations in penile tissue [37]. In our study, the conclusion that l-cysteine elicited H2S-dependent relaxations of murine carotid arteries was confirmed by their sensitivity to AOAA (Fig. 3). Besides, it was shown that AOAA inhibits H2S formation in sheep carotid arteries [26]. Even though the maximal relaxant response of l-cysteine in mice carotid artery was not as high as other arteries such as Aldicarb sale [33], [34], [36] or penile [43], endogenous H2S have an important role to keep the balance of vascular tonus, since AOAA caused a strong contraction (45%) in response to l-cysteine in WT mice carotid arteries (Fig. 3). We should also note that the maximal relaxation response changes according to the tissue type, 15% relaxation to l-cysteine was reported in Wistar Kyoto rat carotid artery as in our current study in mouse carotid artery but 70% in aorta from mouse [34] or rat [36]. The lower magnitude of maximal relaxation to l-cysteine was not related with mouse species since we got 70% relaxation to l-cysteine in mouse penile tissue [43]. eNOS deletion caused an increase in l-cysteine-induced relaxation (Fig. 3). However, exogenous H2S donor NaHS-induced relaxations were not enhanced and even slightly decreased by eNOS deletion and the augmented l-cysteine relaxation in eNOS−/− group was abolished by AOAA or eNOS replacement. Thus we suggest that enhanced relaxation to l-cysteine caused by eNOS deletion is related with an increase in endogenous H2S production rather than downstream mechanism components of H2S mediated relaxation. Abolishment of the relaxation to l-cysteine in WT or eNOS−/− groups and even convertion to contraction by general H2S inhibitor AOAA confirm the role of endogenous H2S. We suggest that increased l-cysteine-induced relaxation may compensate for impaired eNOS-dependent NO-mediated relaxation; which might be clinically important in pathological conditions associated with eNOS dysfunction. Parallel to the current study we have also showed that inhibition of eNOS augments l-cysteine-induced relaxations in mice corpus cavernosum through increasing H2S formation via CSE and MPST expression [43]. Supporting this, CSE and CBS expressions have been found to increase in spontaneous hypertensive rat carotid arteries [35]. While eNOS deletion enhanced l-cysteine-induced relaxation, overexpression of eNOS abolished it. Clearly, one limitation of our study is the lack of a direct assay for H2S production but unfortunately the H2S production by murine carotid arteries is below the detection limit of currently available assays. Pooling tissue from several mice also has the disadvantage of increasing variability. However, we confirmed that the lack of relaxation to l-cysteine in eNOS overexpressing carotid arteries was due to impairment of the endogenous production or bioavailability of H2S, since 1) the relaxation elicited by exogenous H2S (NaHS) administration did not diminish and even increased in eNOS overexpressed carotid arteries, and 2) AOAA did not cause a further inhibition due to readily disrupted H2S synthesis. Another possibility for the abolition of the l-cysteine-induced relaxations may include interaction of NO with H2S to form an inactive nitrosothiol compound to inactivate NO and/or H2S into a less biologically active molecule [9], [44], [45], [46]. However, this would be unlikely in our study since NaHS-induced relaxations were not decreased by eNOS overexpression. Even the relaxation to exogenous H2S was increased and it may be due to a compensatory response to decreased endogenous H2S relaxation and/or formation. The tendency of decrease in relaxation to NaHS in eNOS−/− mice carotid arteries in our study was in accordance with results obtained using aortic rings from eNOS−/− Aldicarb sale mice [11], [15]. However NaHS relaxation has been found to be higher in endothelium denuded cerebral arteries [47]. Therefore, tissue-specific regulation of CSE/H2S by NO can be postulated based on these reports in the literature. Supporting this the regulation of vascular tone by endogenous and exogenous H2S is also tissue specific. A biphasic pattern with contraction at low concentrations and relaxation at higher concentrations was observed in aorta [20], [33], [44] and human internal mammary artery [9] as in the carotid arteries in current study. In contrast, a monophasic relaxation was recorded in the penile vasculature [37]. Despite the fact that we did not assess the molecular mechanisms of the contraction in the murine carotid artery in any detail, our results can eliminate some possibilities suggested before for aorta [44]. It has been suggested that the formation of an intermediate substance resulting from the chemical reaction of NO and H2S could be responsible for the contraction at low concentrations of l-cysteine in rat aorta [45], [46]. However, it is highly unlikely that the latter mechanism can account for the contraction in response to low doses of l-cysteine in murine carotid arteries for the following reasons. If so increasing the amount of NO should cause elevated contraction and decreasing the amount of H2S should lead to decreased contraction. However (1) H2S inhibition by AOAA actually significantly increased l-cysteine-induce contractions. (2) In the presence of higher amount of NO, the contraction was reversed to relaxation upon NaHS stimuli (3 × 10−4 M) and not changed at all in response to l-cysteine in WTxeNOS group.