1 Introduction
NO produced by endothelial cells and stimulating production of cGMP in the underlying smooth muscle cells, is repeatedly proposed to mediate endothelium-dependent vasodilatation(Monica, Bian & Murad, 2016; Shimokawa & Godo, 2020; Vanhoutte, Shimokawa, Feletou & Tang, 2017; Vanhoutte, Zhao, Xu & Leung, 2016). Superoxide anions (O2-) which rapidly bind and inactivate NO, can then cause endothelial dysfunction during conditions of increased oxidative stress that take part in the pathogenesis of cardiovascular diseases (CVD)(Daiber et al., 2017; Forstermann & Munzel, 2006; Zhang, Murugesan, Huang & Cai, 2020). Reported experimental evidence for these widely accepted mechanisms of vasodilatation is frequently incomplete and does not exclude alternative mechanisms. In addition to de novo synthesis, endothelial NO can be released from intracellular stores and can relax vascular smooth muscle independently from cGMP(Batenburg, Garrelds, van Kats, Saxena & Danser, 2004; Batenburg et al., 2004). NO-synthases (especially the nNOS isoform) produce not only NO but also nitroxyl(Fukuto, 2019; Irvine, Ritchie, Favaloro, Andrews, Widdop & Kemp-Harper, 2008; Schmidt, Hofmann, Schindler, Shutenko, Cunningham & Feelisch, 1996; Toda & Okamura, 2003). Like NO, HNO is a potent stimulator of soluble guanylyl cyclase (sGC) but resists inactivation by O2-(Arcaro, Lembo & Tocchetti, 2014; Irvine, Ritchie, Favaloro, Andrews, Widdop & Kemp-Harper, 2008; Leo, Joshi, Hart & Woodman, 2012). In small muscular resistance-sized arteries, the relation between oxidative stress in CVD and endothelium-dependent vasodilatation is even more complex than in large elastic conduit arteries(Ellinsworth, Sandow, Shukla, Liu, Jeremy & Gutterman, 2016; Shimokawa & Morikawa, 2005; Vanhoutte, Shimokawa, Feletou & Tang, 2017). These micro-arteries which control blood pressure and local blood flow, continue to display endothelium-dependent vasodilatation when NOS or sGC are inhibited. In human resistance arteries, this additional pathway may involve H2O2 produced by the microvascular endothelial cells and causing hyperpolarization of the underlying smooth muscle(Ellinsworth, Sandow, Shukla, Liu, Jeremy & Gutterman, 2016; Leurgans et al., 2016; Shimokawa & Morikawa, 2005; Vanhoutte, Shimokawa, Feletou & Tang, 2017). Dismutation of O2- produced by mitochondria, NOS or NADPH oxidases is the main source of this endothelium-derived hyperpolarizing H2O2(Shimokawa & Godo, 2020; Widlansky & Gutterman, 2011; Zhang, Murugesan, Huang & Cai, 2020). In human resistance arteries, increased oxidative stress can thus blunt actions of endothelium-derived NO and promote endothelium-dependent hyperpolarization. The situation is unclear in resistance arteries from patients with resistant CVD requiring surgery. Here, chronic elevation of oxidative stress resulting from multiple risk factors and a long history of CVD might impair the NOS/NO/sGC pathway and promote endothelium-dependent hyperpolarization while chronic treatment with cholesterol lowering, anti-hypertensive and anti-diabetic drugs that have direct and indirect antioxidant properties(Daiber et al., 2017), can protect endothelium-derived NO. Recent observations by Gutterman et al. on endothelium-dependent flow-induced dilatations of human resistance arteries are partly in line with this proposal. In contrast to vessels from patients without clinical signs of coronary artery disease, these dilatations were not inhibited by the non-specific inhibitor of NO-synthases L-NAME but were blocked by scavenging of H2O2 with catalase in vessels isolated from patients undergoing coronary artery bypass grafting(Freed, Beyer, LoGiudice, Hockenberry & Gutterman, 2014; Schulz, Katunaric, Hockenberry, Gutterman & Freed, 2019; Zinkevich, Fancher, Gutterman & Phillips, 2017).
In this study, we tested the hypotheses that endothelium-dependent vasodilatation of resistance arteries from patients with resistant CVD uses mechanisms that are either insensitive to oxidative stress or involve a reactive oxygen species. For this purpose, we isolated resistance arteries from biopsies of parietal pericardium obtained during elective cardiothoracic surgeries. In view of the small size of the arterial tissue samples, we used immunohistochemistry and pharmacological tools to demonstrate presence of mechanistic components and their contribution to the in vitro relaxing effects of the endothelium-dependent vasodilator bradykinin.