Changes in vascular reactivity following administration of isoproterenol for 1 week: a role for endothelial modulation

Abstract

1. The aim of this study was to assess the effects of treatment with isoproterenol (ISO, 0.3 mg kg-1 day-1, s.c.) for 7 days on the vascular reactivity of rat-isolated aortic rings. Additionally, potential mechanisms underlying the changes that involved the endothelial modulation of contractility were investigated. 2. Treatment with ISO induced cardiac hypertrophy without changes in haemodynamic parameters. Aortic rings from ISO-treated rats showed an increase in the contraction response to phenylephrine (PHE) and serotonin, but did not change relaxations produced by acetylcholine or isoproterenol. Removal of the endothelium increased the responses to PHE in both groups. However, this procedure was less effective in ISO-treated as compared with control rats. Endothelial cell removal abolished the increase in the response to PHE in ISO-treated rats. The presence of Nomega-nitro-L-arginine methyl ester shifted the concentration-response curve to PHE to the left in both groups of rats. However, this effect was more pronounced in the ISO group. In addition, aminoguanidine (50 microM) potentiated the actions of PHE only in the ISO group. ISO treatment increased nitric oxide synthase (NOS) activity and neuronal NOS and endothelial NOS protein expression in the aorta. 3. Neither losartan (10 microM) nor indomethacin (10 microM) abolished the effects of ISO on the actions of PHE. Superoxide dismutase (SOD, 150 U ml-1) and L-arginine (5 mM), but neither catalase (300 U ml-1) nor apocynin (100 microM), blocked the effect of ISO treatment. In addition, we observed an increase in superoxide anion levels as measured by ethidium bromide fluorescence and of copper and zinc superoxide dismutase protein expression in ISO-treated rats. 4. In conclusion, our data suggest that ISO treatment alters the endothelial cell-mediated modulation of the contraction to PHE in rat aorta. The increased maximal response of PHE seems to be due to an increase in superoxide anion generation, which inactivates some of the basal NO produced and counteracts NO-mediated negative modulation even in the presence of high NO production and antioxidant defence.

Figure 1

Concentration–response curves to PHE (a), serotonin (b), isoproterenol (c) and acetylcholine (d) in endothelium-intact segments of thoracic aortas from control and isoproterenol-treated rats. Contraction responses are expressed as a percentage of the response to 75 mM KCl and relaxation responses are expressed as percentage of precontraction induced by PHE (∼1 μM). Number of rats is indicated in parentheses. The values are means±s.e.m. 2-way ANOVA; ^*P<0.05 vs Control.

Figure 2

Effects of endothelium removal (E−) on concentration–response curves to PHE in thoracic aortic rings from control (a) and isoproterenol-treated rats (b). Contraction responses are expressed as a percentage of the response to 75 mM KCl. Number of rats is indicated in parentheses. The values are means±s.e.m. 2-way ANOVA, ⁺P<0.05 vs E+. Inset, dAUC to PHE in E− and E+ aortic rings from control and isoproterenol-treated rats; dAUC are expressed as a percentage of the corresponding AUC for E+ aortic rings. Student's t-test, ^*P<0.05 vs Control.

Figure 3

Effects of L-NAME (100 μM) or aminoguanidine (AG, 50 μM) (a and b) or SOD (150 U ml⁻¹) or catalase (CAT, 300 U ml⁻¹) (c and d) or L-arginine (L-arg, 5 mM) (e and f) on concentration–response curves to PHE in intact (E+) thoracic aortic rings from control and isoproterenol-treated rats. Contraction responses are expressed as a percentage of the response to 75 mM KCl. Number of rats is indicated in parentheses. The values are means±s.e.m. Two-way ANOVA, ⁺P<0.05 vs E+. Inset, dAUC to PHE in E+/treatments and E+ aortic rings from control and isoproterenol-treated rats; dAUC are expressed as a percentage of the corresponding AUC for E+ aortic rings. Student's t-test, ^*P<0.05 vs Control.

Figure 4

(a) NOS activity (pmol mg min⁻¹) in isolated aortas from control (CT, n=7) and isoproterenol-treated rats (ISO, n=6) with (E+) and without (E−) endothelium. (b) Top: representative Western blot of nNOS and eNOS protein expression in thoracic aorta of control (CT) and ISO. Left lane, corresponding positive control for each protein (rat brain and human endothelial cells, respectively). Expression of α-actin is also shown as a loading control. Bottom: quantitative analysis of Western blot for eNOS and nNOS protein expression in thoracic aortas of control (CT, n=7) and isoproterenol-treated rats (ISO, n=7). Results (means±s.e.m.) are expressed as the ratio between signal for the NOS protein and signal for α-actin in the corresponding aorta. Student's t-test, ^*P<0.05 vs Control.

Figure 5

(a) Representative fluorescence photomicrographs of microscopic sections of thoracic aorta from control (a, n=3) and isoproterenol-treated rats (b, n=3). Vessels were labelled with the oxidative dye hydroethidine, which produces a red fluorescence when oxidized to ethidium bromide by superoxide anion. AC, adventitial cells; SMC, smooth muscle cells. (b) Representative Western blot (Top) and quantitative analysis (Bottom) for Cu/Zn-SOD protein expression in thoracic aortas of control (CT, n=4) and isoproterenol-treated rats (ISO, n=4). Results (means±s.e.m.) are expressed as the ratio between signal for the Cu/Zn-SOD protein and signal for α-actin in the corresponding aorta. Student's t-test, ^*P<0.05 vs Control.