In Reply:

We thank Kinoshita et al. for their valuable comments on our article regarding helium preconditioning in human endothelium.1  Kinoshita et al. state that there still remain questions regarding the underlying mechanism of the observed endothelial protection after helium-induced late and early preconditioning. In our experiments,1  we have shown significant endothelial dysfunction after 20 min of forearm ischemia induced by inflating a blood pressure cuff. This endothelial dysfunction was prevented by helium early and late preconditioning. The experiments using the nonselective nitric oxide synthase inhibitor, NG-monomethyl-l-arginine, during helium preconditioning were performed to investigate whether endothelial nitric oxide synthase is crucially involved in the mechanism of the observed helium preconditioning. Because co-infusion of NG-monomethyl-l-arginine did not block the protective effect of helium, we concluded that endothelial nitric oxide synthase is not a central mediator of helium-induced preconditioning in this model. In contrast to the reference2  cited by Kinoshita et al., we did not use NG-monomethyl-l-arginine as vasoconstrictive agent during acetylcholine infusion. Therefore, it is correct that we cannot conclude that nitric oxide derived from endothelium is the source of the acetylcholine-induced vasodilatory effect. The dosage of NG-monomethyl-l-arginine, we used equates to that, maximally reduces basal forearm blood flow in healthy volunteers without a possible systemic effect.3  Unfortunately, we were unable to directly obtain endothelial cells from our volunteers to measure oxidative stress in those cells. The last comment raised by the authors pertained to reactive hyperemia directly after release of the blood pressure cuff. As stated in our article, we did not measure reactive hyperemia but performed our measurements in all groups after 20 min of reperfusion. It is, therefore, unlikely that reactive hyperemia caused the observed differences between groups. We agree that further studies are needed to clarify more the mechanisms of helium preconditioning in human endothelium.

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