Reactive oxygen species are involved in regulating alpha1-adrenoceptor-activated vascular smooth muscle contraction

Abstract

Background: Reactive oxygen species (ROS) were shown to mediate aberrant contractility in hypertension, yet the physiological roles of ROS in vascular smooth muscle contraction have remained elusive. This study aimed to examine whether ROS regulate alpha1-adrenoceptor-activated contraction by altering myosin phosphatase activities.

Methods: Using endothelium-denuded rat tail artery (RTA) strips, effects of anti-oxidants on isometric force, ROS production, phosphorylation of the 20-kDa myosin light chain (MLC20), and myosin phosphatase stimulated by alpha1-adrenoceptor agonist phenylephrine were examined.

Results: An antioxidant, N-acetyl-L-cysteine (NAC), and two NADPH oxidase inhibitors, apocynin and VAS2870, dose-dependently inhibited contraction activated by phenylephrine. Phenylephrine stimulated superoxide anion production that was diminished by the pretreatment of apocynin, VAS2870, superoxide scavenger tiron or mitochondria inhibitor rotenone, but not by xanthine oxidase inhibitor allopurinol or cyclooxygenase inhibitor indomethacin. Concurrently, NADPH oxidase activity in RTA homogenates increased within 1 min upon phenylephrine stimulation, sustained for 10 min, and was abolished by the co-treatment with apocynin, but not allopurinol or rotenone. Phenylephrine-induced MLC20 phosphorylation was dose-dependently decreased by apocynin. Furthermore, apocynin inhibited phenylephrine-stimulated RhoA translocation to plasma membrane and phosphorylation of both myosin phosphatase regulatory subunit MYPT1Thr855 and myosin phosphatase inhibitor CPI-17Thr38.

Conclusions: ROS, probably derived from NADPH oxidase and mitochondria, partially regulate alpha1-adrenoceptor-activated smooth muscle contraction by altering myosin phosphatase-mediated MLC20 phosphorylation through both RhoA/Rho kinase- and CPI-17-dependent pathways.

Figure 1

Effects of NAC (a), apocynin (b) and VAS2870 (c) on phenylephrine-induced contraction of RTA strips. Two cumulative concentration-responses were constructed in RTA strips stimulated with α₁-adrenoceptor agonist phenylephrine. For the second contraction, RTA strips were preincubated for 30 min with NAC (10~20 mM), apocynin (0.3~3 mM), VAS2870 (1~10 μM) or vehicle, then were stimulated with agonist in the continuous presence of the inhibitor. Data are mean ± S.E.M. of 6 independent experiments. Force is expressed as the percentage of maximal force obtained in the first contraction.

Figure 2

Phenylephrine-stimulated superoxide anion generation. RTA strips were stimulated with 10 μM phenylephrine and superoxide anion generation was measured by lucigenin-enhanced chemiluminescence as described in Methods. Some vessels were incubated for 1 hr with α₁-adrenoceptor antagonist prazosin (1 μM) or for 30 min with VAS2870 (10 μM), apocynin (1 mM), tiron (10 mM), indomethacin (10 μM), allopurinol (100 μM), rotenone (10 μM) or vehicle (DMSO or ethanol) before and during phenylephrine treatment. As the values obtained in the presence of DMSO or ethanol were not different from that of phenylephrine alone, the data were pooled. Each bar represents mean ± S.E.M. of six to ten independent experiments. *p < 0.001 vs. control; ^#p < 0.05 vs. vehicle.

Figure 3

NADPH oxidase activation in response to phenylephrine stimulation. (a) Time course. RTA strips were stimulated with 10 μM PE for the indicated period. NADPH oxidase activity was measured in RTA homogenates as described in Methods and was expressed as mean ± SEM of 5 independent experiments. *P< 0.05 vs. unstimulated control. (b) Effects of pro-oxidative enzyme inhibitors. RTA strips were incubated for 30 min with the indicated inhibitors, followed by 1-min phenylephrine stimulation. Results were expressed as mean ± S.E.M. of 6~8 independent experiments. *P < 0.05 vs. unstimulated control; #p < 0.01 vs. phenylephrine alone.

Figure 4

Effects of apocynin on phenylephrine-induced MLC₂₀ phosphorylation. RTA strips were pretreated for 30 min with apocynin or vehicle, followed by phenylephrine stimulation. Tissues were snap-frozen at 1 min (a) or 15 min (b). MLC₂₀ phosphorylation was analyzed by 2-D PAGE as described in Methods. Results are expressed as mean ± S.E.M. from 5~9 independent experiments. Apo: apocynin. * p < 0.01 vs. resting control; ^# p< 0.05 vs. phenylephrine alone.

Figure 5

Effects of apocynin on phenylephrine-induced MYPT1 phosphorylation. RTA strips were pre-incubated with apocynin or vehicle for 30 min and then challenged with phenylephrine for 1 or 15 min. Phosphorylation levels of MYPT1 at Thr697 (a) and Thr855 (b) were assessed by immunoblotting using phospho-specific MYPT1 antibody. Equal protein loading was verified by total MYPT1 immunoreactivity. The upper panels show representative immunoblots and the lower panels summarize the densitometric results. Values were normalized against control and data are presented as mean ± S.E.M. of four independent experiments. *p < 0.05 vs. control; #p < 0.05 compared with time-matched phenylephrine alone.

Figure 6

Effects of apocynin on phenylephrine-induced RhoA membrane translocation. RTA strips were pre-incubated with apocynin or vehicle for 30 min and then challenged with phenylephrine for 1 min (a) or 15 min (b). The cytosolic and membrane fractions were separated by ultracentrifugation and RhoA in each fraction were analyzed by immunoblotting as described in Methods. The top panels show representative immunoblots. The bottom panels summarize the quantitative results, expressed as the ratio of membrane RhoA to total RhoA, from six independent experiments. The bars represent means ± S.E.M.. *p < 0.05 vs. control; ^#p < 0.05 vs. phenylephrine alone.

Figure 7

Effects of apocynin on phenylephrine-induced CPI-17 phosphorylation. RTA strips were incubated with apocynin (3 mM) or vehicle for 30 min and then stimulated with phenylephrine for 1 min. The tissue was snap-frozen and CPI-17 phosphorylation was assessed with CPI-17^Thr38 phospho-specific antibody. Equal protein loading was verified by reprobing for total CPI-17 immunoreactivity. The top panel shows the representative blots of phospho-CPI-17^Thr38 and total CPI-17. The bottom panel summarizes the densitometric results. The phosphorylation levels of CPI-17 were quantified as the ratio of phosphorylated CPI-17 to total CPI-17 protein. The bars represent means ± S.E.M. of 9 experiments. *p < 0.05 vs. control; ^#p < 0.05 vs. phenylephrine alone.