2.3 Pharmacological protocols
We compared effects of inhibitors of nitrosative and oxidative mechanisms (see Table 2) on arterial relaxing responses to i) reference agents and to ii) the endothelium-dependent vasodilator bradykinin (BK), during depolarization- and agonist-induced submaximal contraction. The isolated resistance artery segments were first exposed 3 times during 5 min at 20 min interval to 32 mM K+. We previously showed that this stimulus causes a contraction that is ≈40 % of the maximal response to a contractile agonist such as ET-1(Leurgans et al., 2016). In 2 out of 55 experiments, 32 mM K+ resulted in a contraction that was smaller than 0.2 N.m-1 and these experiments were discontinued. During the 3rdK+-induced contraction, the arterial segments were exposed to 1 μM BK for 3 min Following these initial tests, the preparations were incubated for 20 min without (control) or with a pharmacological inhibitor (treated) and relaxing responses to a NO-donor, a nitroxyl-donor compound or exogenous H2O2 were investigated during contraction stimulated with 32 mM K+. Two to five different treatments were always investigated in parallel with one control arterial segment from the same individual patient. After washout of the inhibitor and of the contractile and relaxing stimuli, similar experiments were performed in series evaluating effects of the pharmacological treatments on relaxing responses to BK during contraction stimulated with 32 mM K+. Ultimately, after at least 20 min recovery from this contraction/relaxation cycle, effects of the same pharmacological treatments were tested on relaxing responses to BK during contraction stimulated with ET-1. The concentration of ET-1 was progressively titrated (from 0.063 nM) so that the amplitude of the agonist-induced contraction would match that of the contraction stimulated by 32 mM K+ in the same arterial preparation. In addition to a fair comparison between depolarizing and contractile agonist-induced conditions, this approach allowed to evaluate the effects of the pharmacological inhibitors on the sensitivity of the resistance arteries to ET-1. In initial experiments that evaluated effects of L-NAME, c-PTIO and ODQ, we focused on the maximal response elicited by 1 μM BK. Because an unexpected (lack of) effect was observed, cumulative concentration response curves with half logarithmic steps were constructed for BK (0.01 nM to 1.0 μM) in subsequent experimental series.