Serial sections were equilibrated under identical conditions for 30 min at 37 °C in Krebs–HEPES buffer (in mM: 130 NaCl, 5.6 KCl, 2 CaCl2, 0.24 MgCl2, 8.3 HEPES, and 11 glucose, pH = 7.4). Fresh buffer containing DHE (2 μM) was applied topically to each tissue section, covered with a cover slip, incubated for 30 min in a light-protected humidified chamber at 37 °C, and then viewed with a inverted fluorescence microscope (NIKON Eclipse Ti-S, x40 objective) using the same imaging settings in the untreated and lead-treated rats. Fluorescence

was detected with a 568-nm long-pass filter. For quantification, eight frozen tissue segments per animal were sampled for each experimental condition and averaged. The mean fluorescence densities in the target region were calculated. All values are expressed as the mean ± standard error of the mean (SEM). Contractile responses to phenylephrine were expressed as a percentage of the selleck screening library MEK inhibitor cancer maximal response induced by 75 mM KCl. Vasodilator responses to ACh or SNP were expressed as the percentage of relaxation of the previous contraction. For each concentration–response curve, the maximal effect (Rmax) and the concentration of agonist that produced 50% of the maximal response (log EC50) were calculated using non-linear regression analysis (GraphPad Prism,

GraphPad Software, Inc., San Diego, CA). The sensitivities of the agonists were expressed as pD2 (− log EC50). To compare the effects of L-NAME, TEA, 4-AP, IbTX, ChTX and apamin on the relaxation responses to ACh, some results were expressed as the differences in the area under the concentration–response curves (dAUC) for the control and experimental groups. These values indicate whether the magnitude of the effect of L-NAME, TEA, 4-AP, IbTX, ChTX and apamin is different in the untreated or lead-treated rats. The results were expressed as the mean ± SEM of the number of rats indicated (n). The differences were analyzed using Student’s t-test or two-way ANOVA followed by a Bonferroni test. P < 0.05 was considered to be significant. Lead acetate, l-phenylephrine hydrochloride,

ACh chloride, SNP, sodium pentobarbital, apocynin, SOD, catalase, OUA, L-NAME, TEA, 4-AP, IbTX, CbTX and apamin were purchased from Sigma-Aldrich (St. Louis, USA). The salts and reagents used were of analytical grade from Sigma-Aldrich and Merck (Darmstadt, Germany). Lead exposure Loperamide did not affect the response to KCl (untreated E+: 3.46 ± 0.04 g, n = 38; lead-treated E+: 3.43 ± 0.11 g, n = 40; untreated E−: 3.49 ± 0.03 g, n = 20; lead-treated E−: 3.43 ± 0.09 g, n = 20; P > 0.05). Pre-contraction to phenylephrine used before performing ACh and SNP relaxation curves was similar in the groups (untreated E+: 2.46 ± 0.05 g, n = 10; lead-treated E+: 2.63 ± 0.03 g, n = 10; untreated E−: 2.55 ± 0.11 g, n = 10; lead-treated E−: 2.57 ± 0.04 g, n = 10 P > 0.05). However, this metal reduced vascular reactivity to phenylephrine in the aortic rings (Table 1).