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Am J Physiol Heart Circ Physiol 295: H1935-H1942, 2008. First published September 5, 2008; doi:10.1152/ajpheart.00723.2008
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Inhibition of rat aortic smooth muscle contraction by 2-methoxyestradiol

Yu Gui, Xi-Long Zheng, Jie Zheng, and Michael P. Walsh

Smooth Muscle Research Group, Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, Alberta, Canada

Submitted 14 July 2008 ; accepted in final form 3 September 2008


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Recent studies suggest that 2-methoxyestradiol (2-ME), an estrogen metabolite, has a similar inhibitory effect as 17β-estradiol (E2) on vascular tone. However, it is not known whether 2-ME mediates the effects of E2 or by what mechanism 2-ME regulates smooth muscle contraction. Therefore, we compared the effects of 2-ME and E2 on rat aortic smooth muscle contraction. A preincubation with 2-ME (10 µM) for 1 h inhibited phenylephrine (PE)-induced tension in endothelium-intact, but not -denuded, tissues, whereas E2 inhibited PE-induced contraction in both preparations. The effects of 2-ME and E2 on endothelium-intact preparations were prevented by L-NAME hydrochloride (a nitric oxide synthase inhibitor). The 2-ME treatment reduced PE-induced phosphorylation of the 20-kDa myosin regulatory light chain. The inhibitory effects of 2-ME and E2 were not affected by ICI-182780 (an estrogen receptor antagonist) or actinomycin D (a gene transcription inhibitor); however, the effect of 2-ME, but not E2, was prevented by cycloheximide (a protein synthesis inhibitor). Furthermore, the effect of E2 was not blocked by 1-aminobenzotriazole (a cytochrome P-450 inhibitor) or Ro 41-0960 (a catechol-O-methyltransferase inhibitor). The effect of 2-ME was not mimicked by microtubule-interfering agents (nocodazole or Taxol). We conclude that 2-ME inhibits smooth muscle contractility through an endothelium- and nitric oxide-dependent mechanism, which does not involve estrogen receptors or microtubule disruption. The effect of 2-ME, but not E2, involves de novo protein synthesis. 2-ME does not mediate the inhibitory effect of E2 on smooth muscle contraction. These results support a potentially important role of 2-ME in the regulation of smooth muscle tone in the vasculature.

17β-estradiol

ESTROGEN HAS a variety of effects on the vasculature, including a modification of lipoproteins, an inhibition of atherosclerosis, and a regulation of vascular tone (26). The vasodilatory effects of estrogen can be endothelium dependent or independent and may involve genomic and nongenomic mechanisms (20, 26, 36). In the genomic mechanism, estrogen binds to cytosolic/nuclear estrogen receptors (ERs)-{alpha} and -β, leading to an activation of gene transcription and an increase in endothelial nitric oxide (NO) synthase (eNOS) gene expression, whereas in the nongenomic pathway, estrogen stimulates plasma membrane ERs, leading to an activation of eNOS. Increased eNOS gene expression and activity cause an increase in the production of NO, which in turn induces smooth muscle relaxation mainly via cGMP. 17β-estradiol (E2) at micromolar concentrations inhibits smooth muscle contraction primarily via an endothelium-independent mechanism (4, 14, 16, 32). In addition, estrogen can bind to G protein-coupled receptor 30 (GPR30), a G protein-coupled receptor structurally unrelated to ERs (29, 33). GPR30 is expressed in human blood vessels and in cultured human smooth muscle cells (SMCs) (13), but its role in the vasculature is unclear.

2-Methoxyestradiol (2-ME) is a natural metabolite of endogenous estrogen, produced in various cell types, including vascular SMCs (37), by cytochrome P-450 (CYP450) and catechol-O-methyltransferase (COMT). Plasma concentrations of 2-ME range from picomolar to tens of nanomolar (6). Recently, 2-ME has been used as an anticancer agent at concentrations of >1,000 mg/day in participants of phase II clinical trials (19). Unlike E2, 2-ME has a very low affinity for ERs (23). 2-ME has antiproliferative and proapoptotic effects and, at least in part, mediates the inhibitory effect of E2 on SMC proliferation independent of ERs (5, 38). In vascular SMCs, the 2-ME-induced antiproliferative effect may involve disruption of microtubules (12), which has been implicated in smooth muscle contraction (17, 27, 28). Like E2, 2-ME was found to inhibit high [K+] (64 mM)-induced contraction of rat femoral arterial smooth muscle (16). Recently, 2-ME has been implicated in the control of blood pressure (15).

We therefore investigated the effect of 2-ME on vascular smooth muscle contractility and explored the underlying mechanisms that mediate the effect of 2-ME. We also evaluated the possible involvement of 2-ME in the vasodilatory effects of E2.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. 2-ME, E2, fulvestrant (ICI-182780), actinomycin D (ActD), cycloheximide (CHX), phenylephrine (PE), N{omega}-nitro-L-arginine methyl ester hydrochloride (L-NAME), Taxol, nocodazole, 1-{4-(6-bromobenzo[1,3]dioxol-5-yl)-3a,4,5,9b-tetrahydro-3H-cyclopenta [c]quinolin-8-yl}-ethanone (G-1), 1-aminobenzotriazole (ABT), 2'-fluoro-3,4-dihydroxy-5-nitrobenzophenone (Ro 41-0960), and ACh chloride were purchased from Sigma-Aldrich Canada (Oakville, ON, Canada).

Myography. Tissue preparation was performed as previously described (11). In brief, male and female Sprague-Dawley rats (250–350 g) were cared for in accordance with the recommendations of the Canadian Council on Animal Care and euthanized by decapitation under halothane anesthesia. Animal protocols were approved by the Animal Care Committee of the Faculty of Medicine, University of Calgary. All experiments used male rats, with the exception of Fig. 1D in which the effects of E2 and 2-ME were compared in male and female rats. The aorta was dissected and cut into rings (~2 mm in length), mounted in a myograph chamber (Danish Myo Technology, Skejbyparken, Denmark), and maintained in Krebs solution, bubbled with an 95% O2-5% CO2 gas mixture at 37°C. Isometric tension was recorded using MyoDaq/MyoData 2.1 software (Danish Myo Technology). After equilibration, each ring was contracted with 0.1 µM PE, followed by treatment with 1 µM ACh. Relaxation of >50% to ACh was considered indicative of an intact endothelium. Where applicable, the successful removal of the endothelium was confirmed by the absence of a relaxation response to ACh. The effect of 2-ME on contractility was evaluated by measuring the tension response to PE (0.1 µM) after an incubation with 2-ME for 60 min. Inhibitors or antagonists were added 10 min before the application of 2-ME. After each stimulation, the aortic rings were washed 4 to 5 times. When the tension returned to baseline, the next contractile response was initiated 1 h later.


Figure 1
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  		Fig. 1. The effects of 2-methoxyestradiol (2-ME) and 17β-estradiol (E2) on phenylephrine (PE)-induced contraction of rat aortic smooth muscle. A and B: representative recordings showing the effects of 2-ME (10 µM) and E2 (10 µM) on PE-precontracted rat aortic rings and the effects of prolonged pretreatment with 2-ME (10 µM) and E2 (10 µM) on the PE responses. In controls in which 2-ME or E2 was replaced by vehicle, no significant reduction in tension in response to the third compared with the second PE contraction was observed (see E for cumulative data). C: relaxation (%reduction in tension) induced by 2-ME and E2 added at the peak of PE-induced tension. *P < 0.01, significant relaxant effect (n = 6 replicates). D: summarized data showing the effects of 2-ME and E2 pretreatment on PE-induced contraction of aortic tissues derived from male and female rats (male vs. female, P > 0.05, n = 8 replicates). NS, not significant. E: concentration dependence of the effect of 2-ME pretreatment on PE-induced contraction. The data in the absence of 2-ME indicate the vehicle control. *P < 0.05 vs. PE alone (n = 4 replicates); **P < 0.01 vs. PE alone (n = 4 replicates).

 
Measurement of 20-kDa myosin light chain phosphorylation. Myosin light chain at 20 kDa (LC20) phosphorylation was quantified by Phos-tag SDS-PAGE (31). Briefly, aortic segments were quick frozen in 10% trichloroacetic acid and 10 mM dithiothreitol in acetone and washed with 10 mM dithiothreitol in acetone (35). After lyophilization overnight, tissues were cut into small pieces and protein was extracted with 1 ml of SDS gel sample buffer (18). Equal amounts of protein from each sample, determined by prior quantification of actin following SDS-PAGE and Coomassie blue staining, were loaded on SDS-10% acrylamide gels containing Phos-tag acrylamide and MnCl2 (31). The two-step Western blot analysis procedure was used (31). Anti-LC20 (rabbit polyclonal antibody; Santa Cruz Biotechnology, Santa Cruz, CA) was used as primary antibody and goat anti-(rabbit IgG)-horseradish peroxidase conjugate (Pierce Chemical, Rockford, IL) as secondary antibody. Horseradish peroxidase was detected with the Super-Signal West Femto reagent (Pierce), and the emitted light was detected and quantified with a chemiluminescence imaging analyzer (LAS3000mini; Fujifilm, Tokyo, Japan). Obtained images were analyzed with Multi-Gauge version 3.0 software (Fujifilm). Protein loading levels were normalized by the quantification of calponin by Western blot analysis using polyclonal antibodies raised in rabbits to calponin purified from chicken gizzard; the IgG fraction was purified by adsorption to protein A-Sepharose (34).

Data analysis. Data are presented as either representative recordings or means ± SE. The number of replicates (n) represents the number of isolated aorta preparations used. Differences were evaluated by Student's t-test (paired or unpaired) for comparison of two groups and by ANOVA for comparisons involving three or more groups. P < 0.05 was considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
2-ME inhibits PE-induced contraction. To evaluate whether 2-ME affects smooth muscle contractility, we first examined the effect of 2-ME on PE-precontracted male rat aortic rings. For comparison, the effect of E2 was also examined. In endothelium-intact preparations, PE (0.1 µM) induced robust contraction, which was rapidly reversed by ACh. The application of 2-ME (10 µM) to PE-precontracted tissue did not induce significant relaxation (Fig. 1, A and C), whereas E2 (10 µM) caused partial relaxation (Fig. 1, B and C). E2 and 2-ME relaxed PE-induced contractions by 28 ± 8% (n = 6, P < 0.01) and 2 ± 5%, respectively (Fig. 1C). The relaxation induced by E2 was completely inhibited by the ER antagonist, ICI-182780, as previously reported (7) (data not shown). On the other hand, the preincubation with 2-ME (10 µM) or E2 (10 µM) for 45–60 min resulted in a significant reduction in tension development in response to PE (0.1 µM) (Fig. 1, A and B). PE-induced tension in the presence of 2-ME or E2 was 42 ± 16% and 30 ± 12% of maximal tension, respectively (Fig. 1D). However, the preincubation with vehicle (DMSO or ethanol) did not significantly inhibit subsequent PE-induced tension development (data not shown). The incubation with 2-ME or E2 for 15–30 min did not have the inhibitory effect. The contractile response to PE was completely recovered within 2 h of washout of 2-ME or E2 (data not shown). Similar inhibitory effects of 2-ME and E2 on PE-induced contraction were observed in aortic rings derived from female Sprague-Dawley rats, and no significant differences were observed between female and male rats (Fig. 1D). The inhibitory effect of 2-ME was concentration dependent (Fig. 1E) with significant inhibition observed at 1 µM. Furthermore, a preincubation with 2-ME or E2 for 60 min inhibited KCl (50 mM)-induced tension development to the same extent (51 ± 7% and 46 ± 5% of KCl-induced contraction, respectively, n = 4).

2-ME does not mediate the inhibitory effect of E2 on PE-induced tension. The similarity between the inhibitory effects of 2-ME and E2 pretreatment on PE-induced tension raised the possibility that 2-ME may mediate the effect of E2; i.e., E2 may be metabolized to 2-ME, which inhibits PE-induced contraction. We therefore examined whether ABT (a CYP450 inhibitor) or Ro 41-0960 (a COMT inhibitor), both of which inhibit the conversion of E2 to 2-ME, could block the effect of E2 on PE-induced contraction. As shown in Fig. 2, ABT or Ro 41-0960 did not affect the inhibition by E2. PE-induced tension in the presence of ABT plus E2 and Ro 41-0960 plus E2 was 51 ± 6% and 53 ± 8% of maximal PE-induced tension, respectively (n = 5, P > 0.05 vs. E2 alone).


Figure 2
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  		Fig. 2. The effect of E2 pretreatment on PE-induced contraction of rat aortic rings in the absence and presence of inhibitors of cytochrome P-450 and catechol-O-methyltransferase. 1-Aminobenzotriazole (ABT) (10 µM) and Ro 41-0960 (10 µM) were added 10 min before the application of E2 (n = 5 replicates).

 
The effect of 2-ME does not involve ERs. To investigate whether the inhibitory effect of 2-ME is mediated by ERs, we examined the effect of ICI-182780, an antagonist of ER, on 2-ME-induced inhibition of PE-induced contraction. As shown in Fig. 3, ICI-182780 (100 µM) had no significant effect on 2-ME-induced inhibition of PE-induced contraction. The tension evoked by PE was 43 ± 16% and 50 ± 12% of maximal PE-induced tension in the presence of 2-ME and ICI-182780 plus 2-ME, respectively. Likewise, ICI-182780 did not affect the inhibition of PE-induced tension induced by E2 (Fig. 3). We next investigated whether G-1, an agonist of GPR30, could mimic the effect of 2-ME. After incubation of aortic rings with G-1 (1 µM) for 1 h, PE-induced tension reached 95 ± 6% of maximal tension (n = 6, P > 0.05 vs. DMSO control), indicating that 2-ME does not act via GPR30 (Fig. 3).


Figure 3
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  		Fig. 3. The effects of the estrogen receptor antagonist, ICI-182780, on 2-ME- and E2-mediated inhibition of PE-induced contraction and of the G protein-coupled receptor 30 agonist, 1-{4-(6-bromobenzo[1,3]dioxol-5-yl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinolin-8-yl}-ethanone (G-1), on PE-induced contraction. ICI-182780 (100 µM) and G-1 (1 µM) were added 10 min before the application of 2-ME or E2 (n = 6 replicates).

 
The effect of 2-ME on PE-induced contraction is endothelium and NO dependent. To examine whether the effect of 2-ME is endothelium dependent, we compared its effects on endothelium-intact and -denuded preparations. The incubation with 2-ME markedly reduced PE-induced tension only in endothelium-intact, but not -denuded, aortic rings (Fig. 4A). The inhibitory effect of 2-ME was completely prevented by the NO synthase (NOS) inhibitor L-NAME (10 µM) (Fig. 4A). On the other hand, the preincubation with E2 markedly reduced PE-induced tension in both endothelium-intact and -denuded aortic rings (Fig. 4B). The inhibitory effect of E2 was completely prevented by L-NAME only in the presence of the endothelium (Fig. 4B). In endothelium-intact preparations, the PE-evoked contraction was 39 ± 15%, 112 ± 10%, 28 ± 15%, and 111 ± 18% of maximal PE-induced contraction in the presence of 2-ME, L-NAME plus 2-ME, E2, and L-NAME plus E2, respectively (Fig. 4C). However, in endothelium-denuded preparations, PE-induced tension was 103 ± 6%, 116 ± 13%, 32 ± 17%, and 40 ± 15%, respectively (Fig. 4D).


Figure 4
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  		Fig. 4. The effects of 2-ME and E2 on PE-induced contraction of rat aortic rings with (+EC) and without (–EC) intact endothelium. A and B: representative recordings. C and D: cumulative data. *P < 0.01, PE-induced tension in the presence of 2-ME (or E2) plus N{omega}-nitro-L-arginine methyl ester hydrochloride (L-NAME) is significantly different from that in the presence of 2-ME (or E2) alone (n = 8 replicates); NS (n = 6 replicates, P > 0.05).

 
Inhibition of PE-induced contraction by 2-ME involves a reduction in LC20 phosphorylation. We next evaluated the effect of 2-ME on PE-stimulated phosphorylation of LC20, which initiates smooth muscle contraction (3). Our results showed that 2-ME inhibited phosphorylation of LC20 induced by PE (Fig. 5). The presence of L-NAME completely blocked 2-ME-mediated inhibition of PE-stimulated phosphorylation of LC20 (Fig. 5).


Figure 5
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  		Fig. 5. The effect of 2-ME with and without L-NAME treatment on PE-induced 20-kDa myosin light chain (LC20) phosphorylation in rat aortic rings with intact endothelium. A: phosphorylated LC20 (p-LC20) and unphosphorylated LC20 (LC20) were separated by Phos-tag SDS-PAGE and detected by Western blot analysis with anti-LC20. Comparable tissue protein loading levels were confirmed by Western blot analysis with anti-calponin. B: LC20 phosphorylation levels were quantified as described under METHODS. *P < 0.05 vs. PE alone (n = 4 replicates).

 
The effect of 2-ME involves a translational, but not transcriptional, mechanism. To investigate whether a genomic mechanism is involved in the effects of 2-ME and E2 on PE-induced contraction, ActD (a gene transcriptional inhibitor) and cycloheximide (CHX, a protein synthesis inhibitor) were applied 10 min before the addition of 2-ME or E2. ActD (1 µM) did not affect the inhibition of PE-induced contraction by 2-ME or E2 (Fig. 6). PE-induced tension was 43 ± 13% in the group treated with 2-ME and 29 ± 10% in the group treated with E2 (n = 5, P < 0.01 when compared with DMSO controls). However, preincubation with CHX (10 µM) significantly increased PE-induced tension in the presence of 2-ME (122 ± 16%, n = 5, P < 0.01 vs. 2-ME alone) but did not significantly alter the response in the presence of E2 (38 ± 6%, n = 5, P > 0.05 vs. E2 alone). Therefore, the inhibition of PE-induced contraction by 2-ME, but not E2, may involve de novo protein synthesis.


Figure 6
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  		Fig. 6. The effects of actinomycin D (ActD) and cycloheximide (CHX) on 2-ME and E2 inhibition of PE-induced contraction. Summarized data show the inhibitory effects of 2-ME and E2 on PE-induced contraction in the absence and presence of ActD and CHX. ActD (1 µM) and CHX (10 µM) were added 10 min before the application of 2-ME or E2. *P < 0.01 vs. groups treated with 2-ME alone (n = 5 replicates).

 
The effect of 2-ME is not mimicked by microtubule-interfering agents. To determine whether microtubule disruption mediates the effect of 2-ME, we compared the effects of nocodazole (a microtubule-disrupting agent) and Taxol (a microtubule-stabilizing agent) on PE-induced contraction with that of 2-ME. As shown in Fig. 7, the incubation of aortic rings with either microtubule-interfering agent did not reduce PE-induced tension. In the presence of nocodazole or Taxol, the tension stimulated by PE was 104 ± 18% and 102 ± 8% of maximal PE-induced tension (n = 5, P > 0.05 vs. the DMSO control).


Figure 7
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  		Fig. 7. Nocodazole (ND) and Taxol do not affect PE-induced tension. Rat aortic rings with intact endothelium were preincubated with ND (400 nM) or Taxol (5 µM) for 1 h, followed by the addition of PE. *P < 0.05 vs. the control group treated with DMSO alone (n = 5 replicates).

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In this study, we found that 2-ME shares several properties with E2 with regard to inhibition of vascular smooth muscle contraction: 1) prolonged preincubation of male or female rat aortic rings with 2-ME or E2 markedly reduced the contractile response to PE; 2) neither 2-ME nor E2 acts via estrogen receptors in this context; furthermore, their effects could not be mimicked by the GPR30 agonist, G-1, indicating that this receptor is not involved; and 3) the effects of 2-ME and E2 were unaffected by the inhibition of gene transcription.

There are, however, important distinctions between the effects of 2-ME and E2: 1) 2-ME did not have a significant relaxant effect when added to rat aortic rings precontracted with PE, whereas E2 had a significant (~30%) relaxant effect; 2) the inhibitory effect of 2-ME was blocked by pretreatment with the NOS inhibitor, L-NAME, or by the removal of the endothelium, whereas the E2-induced inhibition was maintained in the absence of the endothelium and blocked by L-NAME only in the presence of the endothelium; and 3) the inhibitory effect of 2-ME, but not E2, required de novo protein synthesis since it was prevented by treatment with CHX. We found that, consistent with distinct properties, the effects of E2 could not be explained by the metabolism to 2-ME since CYP450 or COMT inhibition did not alleviate the effect of E2 on PE-induced contraction. The inhibition of PE-induced contraction following the preincubation with 2-ME could be explained by a reduction in LC20 phosphorylation, which was also prevented by NOS inhibition. It has been previously shown that the inhibitory effect of E2 on high [K+]-induced contraction of the femoral artery and portal vein involved a reduced phosphorylation of LC20 (16). On the other hand, microtubule disruption or stabilization did not affect the contractility, suggesting that an effect of 2-ME on microtubules does not contribute to the inhibition of contraction induced by PE. Based on these observations, we conclude that 2-ME induces protein synthesis related to the eNOS pathway within the endothelium via a mechanism not involving ERs or GPR30. Although the downstream mechanism has not been fully elucidated, it is likely that 2-ME treatment leads to an increased production of NO, which acts on the vascular SMCs to reduce LC20 phosphorylation and inhibit contraction.

Our data with E2 are consistent with previous findings that the treatment of arterial tissues with E2 at micromolar concentrations for 15 min to 2 h caused the inhibition of agonist-induced contraction in both endothelium-denuded and -intact preparations (14, 32). However, in contrast to these earlier reports, our study indicates that E2 can inhibit smooth muscle contraction through two different signaling mechanisms, dependent on whether the endothelium is present or absent. If the endothelium is present, E2 acts on endothelial cells to cause indirectly the inhibition of smooth muscle contraction via a NO-dependent mechanism. On the other hand, if the endothelium is absent, E2 can directly inhibit smooth muscle contraction independent of NO production. Endothelium-dependent and -independent mechanisms have been previously found in E2-induced relaxation of aortic rings (1, 21). Importantly, the present study demonstrated that 2-ME inhibited smooth muscle contraction at concentrations similar to the effective concentrations of E2 on inhibition of contraction as reported by others (10, 16, 21, 32). The inhibitory effect of 2-ME on PE-induced contraction of rat aortic rings was endothelium dependent, which is opposite to the observation in the femoral artery that the inhibition of high [K+]-induced contraction by 2-ME is endothelium independent (16). This apparent discrepancy could be due to different experimental conditions (e.g., method of stimulation) or arterial preparations used in the studies.

Estrogens initiate their functions through genomic and nongenomic mechanisms. We demonstrated that neither the gene transcription inhibitor ActD (1 µM) nor the protein synthesis inhibitor CHX (10 µM) had any effect on E2 inhibition of PE-induced contraction, which is in agreement with the findings of others (16, 30), but inconsistent with the observation that CHX (35 µM) blocked E2 (37 µM for 2 h)-induced inhibition of rat aortic contraction (32). This discrepancy could be due to the different concentrations of CHX used in the studies. However, we found that CHX (10 µM), which did not affect E2-induced inhibition of contraction, was able to prevent the inhibition by 2-ME. These results suggest that the effect of 2-ME, but not that of E2, requires de novo protein synthesis and, therefore, involves the regulation of a translational mechanism.

Nongenomic effects of estrogens may or may not involve ERs (24, 25, 30). Our study showed that the specific estrogen antagonist, ICI-182780, which blocked rapid relaxation of E2 (as described in the RESULTS), had no effect on the inhibition of PE-induced contraction by E2 or 2-ME; and the GPR30 agonist, G-1, did not mimic the effects of E2 or 2-ME. These results suggest that the inhibitory effects of E2 and 2-ME do not involve ERs or GPR30. Our observations differ from previous reports that ERs are involved in estrogen activation of eNOS to increase NO production and induce rapid relaxation (8, 30). However, our observations are consistent with other studies in which supraphysiological concentrations of E2 induced nongenomic relaxation independent of ERs (10, 16). Interestingly, the inhibitory effect of 2-ME on PE-induced contraction is similar to that of E2 in that it does not involve ERs. This could also be due to the very low affinity of 2-ME for ERs.

It has been shown that microtubule-interfering agents, at concentrations of tenths of micromolar, stimulate vascular contraction (17, 27, 28). It is noteworthy that 2-ME, at micromolar concentrations, induces microtubule disruption (2, 9), likely contributing to its antiproliferative effect (12, 22). The fact that 2-ME (10 µM) inhibited rather than enhanced PE-induced contraction of rat aorta suggests that the 2-ME effect is not mediated by microtubule disruption. This was confirmed by the use of microtubule-interfering agents: nocodazole (400 nM) and Taxol (5 µM), which inhibit smooth muscle proliferation (39), did not mimic the inhibitory effect of 2-ME on PE-induced contraction.

Recently, it was reported that levels of COMT and 2-ME are low in women with preeclampsia (15), a condition associated with high blood pressure. The increase in systolic blood pressure observed during late pregnancy in COMT knockout mice or in wild-type mice treated with the COMT inhibitor Ro 41-0960 was prevented by a treatment with 2-ME (15). These observations suggest that 2-ME plays an important role in controlling blood pressure. It is important to note, however, that the effective concentrations of 2-ME in our in vitro experiments are considerably higher than the picomolar to tens of nanomolar concentrations found in normal human plasma (6). The effective concentrations of 2-ME for the inhibition of PE-induced contraction observed in the present in vitro study lie in the micromolar range, i.e., similar to the effective concentrations of estrogen for the inhibition of contraction reported by others (1, 4, 16, 24, 32). These concentrations are also consistent with those required for the 2-ME induction of antiproliferative activity in cultured cells (5, 12, 22, 38). The lipophilic 2-ME might reach higher concentrations within the SMCs than are found in the plasma. Furthermore, SMCs of certain vascular beds (e.g., in kidney and brain) can more effectively metabolize estrogen to 2-ME than others (37).

In conclusion, our results support a role for 2-ME in the regulation of vascular tone and provide insights into the mechanism of action of 2-ME.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by grants from the Heart and Stroke Foundation of Alberta, NWT, and Nunavut (to X.-L. Zheng and M. P. Walsh). Y. Gui is the recipient of a Postdoctoral Fellowship from the Alberta Heritage Foundation for Medical Research (AHFMR). X.-L. Zheng is the recipient of a Senior Scholar Award from AHFMR. M. P. Walsh is the recipient of a Canada Research Chair (tier 1) in Vascular Smooth Muscle Research and an AHFMR Scientist Award.


   ACKNOWLEDGMENTS
 
We are grateful to Dr. Morley Hollenberg for helpful discussions, to Dr. Chris Triggle for providing the myograph system, and to Dr. Kosuke Takeya for advice regarding Phos-tag SDS-PAGE.


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. P. Walsh, Dept. of Biochemistry & Molecular Biology, Faculty of Medicine, Univ. of Calgary, 3330 Hospital Dr. NW, Calgary, Alberta, T2N 4N1 Canada (e-mail: walsh{at}ucalgary.ca)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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