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.