1 nM–300 μM) were determined.
The role of NO in the relaxation induced by ACh was analyzed by incubating the selleck inhibitor vessels with NG-nitro-l-arginine methyl ester (L-NAME, 100 μM, nonspecific NOS inhibitor) for 30 min before phenylephrine or KCl administration. The contribution of K+ channels to ACh-induced relaxation was assessed in aortas previously incubated for 30 min with the K+ channel blockers tetraethylammonium (TEA, 2 mM, nonselective blocker of K+ channels), 4-aminopyridine (4-AP, 5 mM, Kv blocker), iberiotoxin (IbTX, 30 nM, selective BKCa blocker), apamin (0.5 μM, selective blocker of small-conductance Ca2+-activated K+ channels — SKCa) and charybdotoxin (ChTX, 0.1 μM, blocker of KCa and Kv). In some experiments, the concentration–response curves to sodium nitroprusside (SNP, 0.01 nM–0.3 μM) were performed Selleckchem BMS 354825 in segments contracted with phenylephrine (1 μM). The role of the Kv and BKCa channels in the SNP-induced
relaxation was analyzed by incubating the vessels with 4-AP and IbTX, respectively, for 30 min before phenylephrine administration. The influence of the endothelium on the response to SNP in untreated and lead-treated rats was investigated after its mechanical removal, which was performed by rubbing the lumen with a needle. The absence of endothelium was confirmed by the inability of 10 μM acetylcholine (ACh) to produce relaxation. The functional activity of the Na±/K+-ATPase was measured in segments from untreated and lead-treated rats using K+-induced relaxation, as described by Weeb and Bohr (1978) and modified by Rossoni et al. (2002). After a 30-min equilibration period in normal Krebs, the preparations were incubated for 30 min in K+-free Krebs. The vessels were subsequently pre-contracted with
phenylephrine, and once a plateau was attained, the KCl concentration was increased stepwise (1, 2, 5 and 10 mM) with each step lasting for 2.5 min. After a washout period, the preparations were incubated next with 100 μM ouabain (OUA) for 30 min to inhibit sodium pump activity, and the K+-induced relaxation curve was repeated. To study the involvement of NO, inducible NO synthase (iNOS) and K+ channels in OUA-sensitive Na+K+-ATPase functional activity, the rings were incubated with L-NAME (100 μM), aminoguanidine (50 μM) and TEA (2 mM), respectively. Moreover, the influence of the endothelium was investigated, repeating the same protocols after its mechanical removal. The oxidative fluorescent dye dihydroethidium (DHE) was used to evaluate O2− production in situ, as previously described by Wiggers et al. (2008). Hydroethidine freely permeates cells and is oxidized in the presence of O2− to ethidium bromide, which is trapped by intercalation with DNA. Ethidium bromide is excited at 546 nm and has an emission spectrum of 610 nm.