AJP - Regu Add DOIs to your references at manuscript stage!
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Regul Integr Comp Physiol 287: R342-R348, 2004. First published May 6, 2004; doi:10.1152/ajpregu.00156.2004
0363-6119/04 $5.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
287/2/R342    most recent
00156.2004v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (13)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Budzyn, K.
Right arrow Articles by Sobey, C. G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Budzyn, K.
Right arrow Articles by Sobey, C. G.

NEUROHUMORAL CONTROL OF CARDIOVASCULAR FUNCTION

Chronic mevastatin modulates receptor-dependent vascular contraction in eNOS-deficient mice

Klaudia Budzyn, Philip D. Marley, and Christopher G. Sobey

Department of Pharmacology, The University of Melbourne, Parkville, Victoria 3010, Australia

Submitted 10 March 2004 ; accepted in final form 22 April 2004


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
We tested the hypothesis that endothelial nitric oxide (NO) synthase (eNOS)-derived NO modulates rho-kinase-mediated vascular contraction. Because 3-hydroxy-3-methylglutaryl (HMG)-CoA-reductase inhibition can both upregulate eNOS expression and inhibit rhoA/rho-kinase function, a second hypothesis tested was that statin treatment modulates rho-kinase-mediated contraction and that this can occur independently of eNOS. Contractile responses to the receptor-dependent agonists serotonin and phenylephrine but not to the receptor-independent agent KCl were greater in aortic rings from eNOS-null (eNOS–/–) vs. wild-type (eNOS+/+) mice. Similarly enhanced responses were seen in eNOS+/+ rings after acute NOS inhibition. The rho-kinase inhibitor Y-27632 abolished or profoundly attenuated responses to receptor agonists in both eNOS+/+ and eNOS–/– rings, but responses in eNOS+/+ were more sensitive to Y-27632. Mevastatin treatment (20 mg/kg sc per day, 14 days) reduced responses to serotonin and phenylephrine in female mice of both strains. KCl-induced contractions were slightly smaller in eNOS+/+-derived aortic rings only. Levels of plasma cholesterol, and aortic expression of rhoA and rho-kinase, did not differ between groups. Thus eNOS-derived NO suppresses rhoA/rho-kinase-mediated vascular contraction. Moreover, a similar suppressive effect on rho-kinase-mediated vasoconstriction by statin therapy occurs independently of effects on eNOS or plasma cholesterol.

endothelium; vasoconstriction; statins

THE CA2+-INDEPENDENT increase in vascular smooth muscle tone that can occur through inhibition of myosin light-chain phosphatase (MLCP) is known as Ca2+ sensitization and appears to be largely mediated by activation of the small GTPase rhoA and its downstream effector rho-kinase (35, 37). RhoA may be activated by several signaling pathways, including the binding of G protein-coupled receptor (GPCR) agonists (35). RhoA-activated rho-kinase then phosphorylates and inhibits MLCP (16), leading to vasoconstriction. By contrast, nitric oxide (NO) contributes to regulation of vascular tone by relaxing smooth muscle. NO is formed within vascular endothelium by the endothelial isoform of NO synthase (eNOS) and is the predominant mediator of endothelium-dependent vascular relaxation (29). Thus the rhoA/rho-kinase and eNOS/NO signaling pathways essentially have major opposing roles in the vasculature, although it is poorly understood whether these mechanisms normally interact.

It is well established that bioactivity of endothelium-derived NO is commonly diminished in many cardiovascular disease states (10). Conversely, several studies have now reported that vascular rhoA/rho-kinase activity is elevated in cardiovascular disease states such as hypertension, arteriosclerosis, and cerebral and coronary vasospasm (34). To our knowledge, however, the possibility that vascular rho-kinase function is augmented as a consequence of chronically reduced eNOS-derived NO activity has not yet been directly tested.

3-Hydroxy-3-methylglutaryl (HMG)-CoA-reductase inhibitors, or "statins," are widely used clinically to lower elevated blood cholesterol levels. Indeed, the beneficial effects of statin therapy appear to extend far beyond their cholesterol-lowering abilities (36). Experimental use of statins has also been reported to confer cholesterol-independent protection against stroke, improve endothelial function, and decrease platelet aggregation (9, 22). These effects have been attributed to upregulation of eNOS expression and consequently increased NO production, due to inhibition of rhoA isoprenylation and subsequently increased eNOS mRNA stability via effects on the endothelial actin cytoskeleton (21, 24). Although, in theory, statins could also modulate vascular contractility through interference with isoprene formation and subsequent rhoA/rho-kinase signaling in vascular smooth muscle, independently of any effect on eNOS/NO, no study has so far tested this hypothesis. First, we therefore tested whether rho-kinase-mediated vascular contraction is enhanced in the chronic and/or acute absence of NO. Second, we tested whether statin treatment can suppress rho-kinase-mediated vascular contraction, and if so, whether this can occur independently of effects on NO activity and plasma cholesterol levels.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
General procedures. Adult male and female mice of eNOS+/+ (wild type, C57BL/6J) and eNOS mutant (eNOS–/– on a C57BL/6J background; Jackson Laboratory, stock no. 002684, colony maintained by homozygous sibling matings) genotype were studied (body wt 25 ± 1 g, mean ± SE; n = 121). Thoracic aortic rings (3–4 mm) were mounted at 5 mN in a 5-ml chamber of a four-channel myograph (model 610M, MultiMyograph) containing Krebs-bicarbonate solution bubbled with 5% CO2 in O2 at 37°C. Tension was continuously recorded on a chart recorder (model 3721, Yokogawa, Japan). Each ring was first exposed to an isotonic high-K+-containing physiological saline solution (KPSS), in which Na+ in Krebs solution was replaced by K+ ([K+]KPSS = 124 mM). The KPSS-induced contraction was allowed to achieve a stable level over 30–40 min. After several washouts and return to stable baseline (~5 mN), each ring was precontracted to ~50% of its KPSS response with either serotonin (0.03–0.1 µM) or phenylephrine (0.1–0.3 µM). Sustained relaxation (>70% of precontracted tone) of eNOS+/+ rings in response to ACh (10 µM) confirmed the endothelium to be functionally intact. By contrast, the lack of such relaxation to ACh of eNOS–/–-derived rings phenotypically confirmed the absence of eNOS expression. After several washouts and return to stable baseline, concentration-response curves were established for the vascoconstrictor agents serotonin, phenylephrine, or KCl. One or two such curves were typically performed per ring.

Effects of rho-kinase and NOS inhibition on vascular contraction. The effect of the selective rho-kinase inhibitor Y-27632 (1–10 µM) (7, 13) on vasoconstrictor responses was assessed by treating aortic rings for 30 min before commencing cumulative additions of a contractile agent. Similarly, the effect of acute NOS inhibition by the isoform nonselective NOS inhibitor NG-nitro-L-arginine methyl ester (L-NAME, 100 µM) was assessed after 30-min treatment before addition of constrictors. During both sets of experiments, vehicle (0.9% saline)-treated rings served as controls. In other experiments, the contractile response to 100 µM L-NAME after 2 h was assessed as an estimate of basal NO activity in eNOS+/+-derived rings precontracted to ~20% of the KPSS response with one of the three contractile agonists. The lack of response of eNOS–/– rings to L-NAME provided further confirmation of the eNOS–/– genotype.

Chronic mevastatin treatment. Mevastatin (Calbiochem, La Jolla, CA) was activated by alkaline hydrolysis and then neutralized under sterile conditions. This solution was then dried down by vacuum centrifugation and reconstituted in saline at 33 mg/ml. Aliquots of this preparation were stored at –20°C for up to 2 mo before use. Female mice of either strain were treated with vehicle (0.9% saline, n = 3) or mevastatin (20 mg/kg sc per day, n = 11) for 14 days via a surgically implanted osmotic minipump (Alzet model 2002, Alza). Minipump implantation was performed aseptically under brief anesthesia (methohexitone 65 mg/kg ip).

Western blotting. Total rhoA and rho-kinase in mouse aorta were measured by Western blotting using antisera to rhoA (Santa Cruz Biotechnology sc-179, San Diego) or rho-kinase [raised against a GST fusion protein of the rho-kinase (ROK{alpha}) coiled coil domain, residues 429–968, expressed in E. coli from a pGEX plasmid provided by Dr. T. Leung (3)].

Analyses and statistics. All responses to contractile agents are presented as percentage of the KPSS response of that aortic ring. Concentration-response data for serotonin and phenylephrine were fitted to a Sigmoid plot using GraphPad Prism version 3.0a, which estimated the pEC50 value. KCl curves were generally found to be nonsigmoidal, and so pEC50 values were not calculated. Relaxation responses are presented as %inhibition of the precontracted level of tone. Each n represents the number of animals used. Statistical analysis was carried out using Student's paired or unpaired t-test or a one-way ANOVA followed by Newman-Keuls multiple comparisons test (as appropriate). P < 0.05 was considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
General data. KPSS-induced vascular contractions were slightly greater in eNOS+/+ vs. eNOS–/– aortic rings (11.4 ± 0.05 vs. 10.3 ± 0.4 mN, n = 57 and 53, respectively; P < 0.05). In rings from eNOS+/+ mice, ACh elicited a large, sustained relaxant response (79 ± 1%, n = 57), whereas in rings from eNOS–/– mice ACh elicited a large, transient contraction (82 ± 6%, n = 53; P < 0.05 vs. eNOS+/+).

Effect of eNOS expression on responses to contractile agents. Serotonin, phenylephrine, and KCl each caused concentration-dependent contraction of aortic rings from both eNOS+/+ and eNOS–/– mice (Fig. 1, A, C, and E). Maximum responses to the receptor agonists serotonin and phenylephrine were greater in aortic rings from eNOS–/– mice than in rings from eNOS+/+ mice (P < 0.05, Fig. 1, A and C, respectively). However, maximum responses to the nonreceptor contractile agent KCl did not differ between strains (Fig. 1E). pEC50 values were significantly lower (~2-fold) in eNOS–/– vs. eNOS+/+ rings for serotonin and phenylephrine (Table 1). Interestingly, maximum contractile responses to serotonin were greater in aortic rings from male vs. female eNOS+/+ (129 ± 5 vs. 102 ± 7%, n = 10–12; P < 0.05), but not eNOS–/– (161 ± 6 vs. 150 ± 6%, n = 10–12; P > 0.05) mice. There was no such gender difference in responses to phenylephrine or KCl in either eNOS+/+ or eNOS–/– mice (data not shown).



View larger version (32K):
[in this window]
[in a new window]
  		Fig. 1. Effects of chronic and acute nitric oxide (NO) deficiency. Left: concentration-response curves obtained in aortic rings from endothelial nitric oxide synthase (eNOS)-null (eNOS–/–) and wild-type (eNOS+/+) mice in response to serotonin (5-HT, both n = 22; A), phenylephrine (Phe, n = 18 and 11; C), and KCl (both n = 15; E). Right: comparative data from paired rings of eNOS+/+ aorta treated with vehicle or NG-nitro-L-arginine methyl ester (L-NAME), showing concentration-response curves to 5-HT (n = 6; B), Phe (n = 5; D), and KCl (n = 5; F). Maximum responses to 5-HT and Phe were augmented by either chronic or acute NO deficiency (*P < 0.05 vs. eNOS–/– or L-NAME treatment, respectively). KPSS, high-K+-containing physiological saline solution.

 

View this table:
[in this window]
[in a new window]
  		Table 1. pEC50 values for serotonin and phenylephrine concentration-response data

 
Effect of acute NOS inhibition. In eNOS+/+aortic rings treated with L-NAME, responses to serotonin, phenylephrine, and KCl resembled those of control rings from eNOS–/– mice (Fig. 1, B, D, and F; compare with Fig. 1, A, C, and E). L-NAME had no effect on contractile responses to any agent in eNOS–/– aortic rings (data not shown). After precontraction with serotonin, vascular contractions in response to L-NAME were smaller in male vs. female eNOS+/+ aortic rings (45 ± 8 vs. 75 ± 6%, n = 7–9; P < 0.05). However, no such gender difference was observed in response to L-NAME when eNOS+/+ aortic rings were precontracted with KCl (61 ± 17 vs. 59 ± 12%, n = 6–8; P > 0.05) or phenylephrine (154 ± 33 vs. 99 ± 5%, n = 2–3).

Effect of rho-kinase inhibition on contractile responses. Maximum responses to serotonin and phenylephrine were profoundly inhibited in a concentration-dependent manner by the selective rho-kinase inhibitor Y-27632 (1–10 µM) in aortic rings from both eNOS+/+ and eNOS–/– mice (Fig. 2, A–D). Inhibitory effects of Y-27632 were more pronounced in aortic rings from eNOS+/+ than eNOS–/– mice. By contrast, although slightly reduced by Y-27632, maximum responses to KCl were relatively resistant to effects of rho-kinase inhibition in eNOS+/+ and eNOS–/– aortic rings (Fig. 2, E and F, respectively). Effects of Y-27632 on pEC50 values, if any, were modest (Table 1).



View larger version (33K):
[in this window]
[in a new window]
  		Fig. 2. Effects of rho-kinase inhibition. Concentration-response curves obtained in aortic rings from eNOS+/+ (left) and eNOS–/– mice (right) in response to serotonin (5-HT; A and B), Phe (C and D), and KCl (E and F); n = 5–12 in all groups. Responses of rings treated with 1 or 10 µM Y-27632 are compared with those of vehicle-treated (0 µM Y-27632) rings. *Maximum response differs from vehicle-treated rings, P < 0.05.

 
Effects of chronic mevastatin treatment. Plasma cholesterol concentration was not altered by mevastatin treatment (control eNOS+/+ = 1.28 ± 0.08 mM, n = 8; mevastatin-treated eNOS+/+ = 1.31 ± 0.19 mM, n = 6; control eNOS–/– = 1.53 ± 0.17 mM, n = 6; mevastatin-treated eNOS–/– = 1.57 ± 0.24 mM, n = 5). Mevastatin treatment resulted in 20–30% smaller responses to KPSS in both eNOS+/+ (control = 11.0 ± 0.6 mN, n = 29; mevastatin-treated = 8.7 ± 0.8 mN, n = 6; P > 0.05) and eNOS–/– (control = 10.3 ± 0.5 mN, n = 20; mevastatin-treated = 6.8 ± 0.2 mN, n = 5; P < 0.05) mice. There were no differences in responses to ACh between aortic rings from control and mevastatin-treated mice (control eNOS+/+ = 80 ± 2%, mevastatin-treated eNOS+/+ = 81 ± 4%; control eNOS–/– = –88 ± 9%, mevastatin-treated eNOS–/– = –80 ± 2%).

Mevastatin treatment reduced maximum responses to serotonin and phenylephrine in aortic rings from both eNOS+/+ and eNOS–/– mice (Fig. 3, A–D). pEC50 values for serotonin were decreased slightly in aortic rings from eNOS+/+ mice and increased slightly in rings from eNOS–/– mice (Table 1). pEC50 values for phenylephrine were higher in aortic rings from eNOS–/– mice after mevastatin treatment (Table 1).



View larger version (28K):
[in this window]
[in a new window]
  		Fig. 3. Effects of chronic mevastatin treatment. Concentration-response curves obtained in aortic rings from eNOS+/+ (left) and eNOS–/– mice (right) in response to serotonin (5-HT; A and B), Phe (C and D), and KCl (E and F); n = 5–10 in all groups. Responses of rings from mice treated with vehicle or mevastatin are compared. *Maximum response differs from rings from control mice, P < 0.05.

 
Maximum responses of eNOS+/+ aortic rings to KCl were slightly but significantly reduced by mevastatin (P < 0.05, Fig. 3E). However, mevastatin treatment had no effect on responses to KCl in aortic rings from eNOS–/– mice (Fig. 3F).

Contractions in response to L-NAME (100 µM) of aortic rings precontracted with serotonin tended to be smaller in rings from mevastatin-treated eNOS+/+ mice (48 ± 11%, n = 6) than in those from untreated eNOS+/+ mice (75 ± 6%, n = 9; P = 0.08).

Effect of chronic mevastatin treatment on rho-kinase expression in mouse thoracic aorta. Western blot analysis revealed no effect of chronic mevastatin treatment on rhoA (data not shown) or rho-kinase expression in thoracic aorta of both eNOS+/+ and eNOS–/– mice (Fig. 4).



View larger version (26K):
[in this window]
[in a new window]
  		Fig. 4. Expression of rho-kinase after chronic mevastatin treatment. Open bars, control. Filled bars, mevastatin treated. Left to right: bars correspond to lanes of Western blot shown above. Top: representative Western blot showing rho-kinase expression in thoracic aorta of control or mevastatin-treated eNOS+/+ and eNOS–/– mice. Bottom: densitometric data are summarized; n = 4–5 in each group.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The present study is the first to directly investigate the importance of eNOS expression in modulation of rho-kinase-mediated vascular contraction after chronic statin treatment. There are several important findings of this study. First, vascular contraction in response to the GPCR agonists serotonin and phenylephrine was selectively greater in aortic rings from eNOS–/– vs. eNOS+/+ mice. Second, acute inhibition of NO synthesis markedly augmented responses of eNOS+/+ aortic rings to these agonists, to become functionally identical to those of eNOS–/– rings. Thus both acute and chronic NO deficiency lead to augmented vascular contractility to serotonin and phenylephrine. Third, vascular responses to serotonin and phenylephrine were profoundly attenuated by the rho-kinase inhibitor Y-27632 in both eNOS+/+- and eNOS–/–-derived aortic rings. However, responses in rings from eNOS+/+ mice were considerably more sensitive to inhibition by Y-27632. Decreased sensitivity of GPCR agonist responses to rho-kinase inhibition during NO deficiency is consistent with the concept that NO normally inhibits vascular rhoA/rho-kinase function. Fourth, chronic mevastatin treatment impaired vascular responses of female eNOS+/+- and eNOS–/–-derived aortic rings to serotonin and phenylephrine without affecting plasma cholesterol concentration. Thus statins modulated vascular function independently of increased endothelial NO synthesis or reduced plasma cholesterol and conceivably acted by interfering with the activity of the rhoA/rho-kinase signaling pathway, which was found to be essential for contraction by these two agonists in mouse aorta.

Physiological roles of vascular NO. NO is formed within the vascular endothelium by eNOS and modulates vascular tone by mediating endothelium-dependent relaxation in response to certain vasodilators such as ACh (29). It has been known for some time that endothelium-derived NO can also modulate responses to numerous vasoconstrictor stimuli, including serotonin and {alpha}-adrenoceptor agonists (6). Thus NO removal either by endothelial denudation (6, 20) or pharmacological block of NO production (2, 29) leads to enhanced vasoconstrictor responses. However, because highly selective eNOS inhibitors are not yet available, pharmacological investigation of specific role(s) of eNOS have been restricted. The recent availability of genetically manipulated animals lacking the eNOS gene has enabled a more definitive means for assessing the importance of endothelial NO in vascular function (12, 19).

In the present study, serotonin- and phenylephrine-induced contractile responses of aortic rings from eNOS–/– mice were greater than those from eNOS+/+ mice. This is consistent with findings in aorta (17) and femoral (39) and carotid arteries (19) of eNOS–/– mice and suggests that genetically based chronic deficiency of eNOS-derived NO leads to augmented vascular contractility, particularly to GPCR agonists. Despite its lack of isoform selectivity, we used the NOS inhibitor L-NAME to investigate whether acute NO deficiency in normal (eNOS+/+) mice could lead to similarly augmented vascular contractions as those observed in aortas from eNOS–/– mice. Indeed, responses were greater in aortas from eNOS+/+ mice after L-NAME treatment, becoming phenotypically identical to those from eNOS–/– mice and indicating that both chronic and acute NO deficiency equally lead to augmented contractility. Importantly also, L-NAME treatment had no effect on responses of aortic rings from eNOS–/– mice, showing that other sources of NO (e.g., inducible NOS and neuronal NOS) do not typically affect vascular contractility to these agents in mouse aorta.

Interactions between rhoA/rho-kinase and NO. The small G protein rhoA and its downstream effector rho-kinase contribute to agonist-induced vascular smooth muscle contraction via Ca2+ sensitization (11, 37). Both the GPCR agonists serotonin and phenylephrine have been reported to elicit vascular contraction via this pathway (30, 37). Recent data suggest that there may be several levels of interaction between the rhoA/rho-kinase signaling pathway and eNOS/NO within the vasculature. First, rhoA is reported to downregulate eNOS expression by decreasing its mRNA stability in cultured endothelial cells, possibly through effects on the cellular actin cytoskeleton (21, 25). Several other studies (26, 32, 40) have shown that both cGMP (which is stimulated by NO) and its downstream effector, cGMP-dependent protein kinase, can functionally antagonize Ca2+ sensitization by activating MLCP or inhibiting rhoA, respectively. Thus there is evidence that both the NO/cGMP and rhoA/rho-kinase signaling pathways can each suppress the function of the other. We therefore speculated that the augmented contractility to serotonin and phenylephrine in aorta from eNOS–/– mice is due to rho-kinase function being augmented in the absence of opposition from endothelium-derived NO. Our data, which showed that Y-27632 abolished or profoundly inhibited contractile responses to serotonin and phenylephrine in eNOS–/– mice, is consistent with this proposal.

Rho-kinase and NO function in vascular disease. Several recent reports have provided strong evidence that vascular rhoA/rho-kinase activity is elevated in disease states such as hypertension (5, 27, 33, 37), cerebral (31) and coronary (15) vasospasm, arterial lesion formation (28), and vascular hypertrophy (41). This phenomenon therefore appears to occur in parallel with the widely accepted characteristic of diminished levels of endothelium-derived NO in such vascular diseases (10). Thus, given that activated rhoA can reportedly inhibit eNOS expression (24), excessive stimulation of the procontractile rhoA/rho-kinase pathway could exacerbate vascular dysfunction, in part by reduction of eNOS expression. On the other hand, and consistent with the present findings, is the concept that NO deficiency may result in increased rho-kinase-mediated Ca2+ sensitization in vascular smooth muscle due to lack of rhoA/rho-kinase suppression by endothelial NO. Thus elevated rho-kinase activity in the absence of NO would be expected to require higher concentrations of a rho-kinase inhibitor to normalize or attenuate exaggerated contractile responses to certain agonists, as was observed here. Indeed, although we found responses of aortic rings from both mouse strains to serotonin and phenylephrine to be dependent on rho-kinase activity, GPCR responses of eNOS+/+-derived rings were much more sensitive to rho-kinase inhibition than were eNOS–/– vessels. By contrast, however, responses of both strains to KCl were relatively unaffected by Y-27632, with maximum KCl contractions remaining ≥85% of the KPSS response in the presence of 10 µM Y-27632. This may indicate that in both strains, rho-kinase substantially (and probably completely) mediates aortic contractile response to these receptor agonists, but not to KCl, as predicted. It is unlikely that the modest effects of Y-27632 on KCl responses were actually due to inhibition of a component of contraction caused by norepinephrine released during depolarization of adventitial nerves, and thus mediated by activation of {alpha}-adrenoceptors and rhoA/rho-kinase, because similar effects were observed in the presence of the {alpha}1-adrenoceptors antagonist prazosin (data not shown). As mentioned, our findings support the concept that responses to serotonin and phenylephrine in eNOS–/– vessels are indeed augmented due to increased activity of rhoA/rho-kinase in the absence of NO. Another recent study in rat aortic segments either endothelium denuded or treated with a NOS inhibitor also concluded that NO can oppose rho-kinase-mediated contractions and that there is greater sensitivity to rho-kinase inhibition by Y-27632 (and therefore perhaps less rho-kinase activity) when NO is present (4).

Statin therapy. Besides lowering elevated plasma cholesterol levels, statins are now known to confer several other benefits for cardiovascular function, including stabilization of atherosclerotic plaques (36), limiting cerebral infarct size (1, 9, 22), and reducing oxidative stress (8, 38). Thus far, such beneficial effects of statins have been thought to be solely due to their ability to increase eNOS expression and activity and hence increase NO bioavailability in the vasculature (14, 18, 23). The current concept of how this occurs is that by blocking the isoprenylation of rhoA in endothelium, statins prevent its ability to translocate from the cytosol and embed in the membrane, thereby preventing its activation by binding GTP in exchange for GDP (24, 36). In this way, prevention of the isoprenylation of rhoA results in disinhibition of eNOS expression by increasing eNOS mRNA stability (21, 24). Theoretically, however, statins may be equally able to modulate vascular contraction through direct interference with rhoA/rho-kinase signaling in vascular smooth muscle, independently of any additional indirect effects on eNOS function.

Using eNOS–/– mice, we directly investigated the importance of eNOS expression in modulation of rho-kinase-mediated vascular contraction after chronic statin treatment. Indeed, chronic mevastatin treatment suppressed contractile responses of aortic rings from both eNOS+/+ and eNOS–/– female mice to serotonin and phenylephrine, indicating that such an effect of statins can occur independently of NO. The present dose of mevastatin did not affect plasma cholesterol levels, as reported previously (1, 9). Also noteworthy was the relative lack of effect of mevastatin treatment on KCl-induced contractile responses, analogous to the selectivity of rho-kinase inhibition in attenuating contractions to serotonin and phenylephrine. Thus the data are consistent with the effect of mevastatin occurring via interference with rhoA/rho-kinase signaling in vascular smooth muscle. In any case, it is apparent that even in eNOS+/+ mice, mevastatin treatment probably did not substantially increase levels of endothelial NO release because neither ACh-induced relaxation nor L-NAME-induced contraction were enhanced. These effects of mevastatin were found to occur without any change in total rhoA or rho-kinase expression, although it is possible that the level of activation or membrane association of these proteins was modulated by the treatment.

Gender difference in NO activity. We noted a gender difference in contractile responses to serotonin (but not phenylephrine or KCl) in aortic rings from eNOS+/+ but not eNOS–/– mice, with responses being greater in males than in females. As mentioned, vascular contractions induced by serotonin can be modulated in some vessels by the simultaneous release of NO via activation of endothelial serotonergic receptors (6). A similar gender difference in response to serotonin was recently reported in mouse carotid artery (19). This effect could be due to estrogen-dependent upregulation of endothelial serotonergic receptors or their subsequent signaling to eNOS, to increase NO release in response to serotonin in females, as opposed to any general effect of estrogen on endothelial NO synthesis. This notion that serotonin-induced NO release from eNOS is greater in females than males is further supported by our observations that 1) contractions to L-NAME after serotonin (but not phenylephrine or KCl) precontraction were greater in females than in males; 2) there was no gender difference in responses to serotonin in eNOS–/– mice; 3) there was no gender difference in contractile responses to phenylephrine or KCl in either strain; and 4) there was no gender difference in the relaxant response to ACh in eNOS+/+ mice. Thus this gender-related difference in response to serotonin is clearly both agonist and eNOS related.

The results of this study demonstrate that both chronic and acute NO deficiency lead to augmented contractility of the mouse aorta, particularly in response to two GPCR agonists. Furthermore, sensitivity to rho-kinase inhibition is decreased in the absence of endothelial NO production. Taken together, these observations strongly suggest a novel physiological role for NO in specifically opposing vascular contraction occurring through GPCR-related rho-kinase-mediated Ca2+ sensitization. This study has also shown that chronic mevastatin treatment can suppress vascular contractile responses dependent on rho-kinase. This effect of statin treatment can occur independently of eNOS/NO, suggesting a more direct effect perhaps by disrupting rhoA/rho-kinase signaling in vascular smooth muscle. Given that excessive activity of the rhoA/rho-kinase pathway has been implicated in the pathology of a range of vascular disorders, our data support the notion that this could be the direct result of decreased NO bioactivity and are consistent with the observed cholesterol-independent clinically beneficial effects of statins in such conditions.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
These studies were supported by a Project Grant from the National Health and Medical Research Council of Australia (208969).


   FOOTNOTES
 

Address for reprint requests and other correspondence: C. G. Sobey, Dept. of Pharmacology, The Univ. of Melbourne, Grattan St., Parkville, Victoria 3010, Australia (E-mail: cgsobey{at}unimelb.edu.au).

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
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Amin-Hanjani S, Stagliano NE, Yamada M, Huang PL, Liao JK, and Moskowitz MA. Mevastatin, an HMG-CoA reductase inhibitor, reduces stroke damage and upregulates endothelial nitric oxide synthase in mice. Stroke 32: 980–986, 2001.[Abstract/Free Full Text]
  2. Carter RW, Begaye M, and Kanagy NL. Acute and chronic NOS inhibition enhances {alpha}2-adrenoreceptor-stimulated RhoA and Rho kinase in rat aorta. Am J Physiol Heart Circ Physiol 283: H1361–H1369, 2002.[Abstract/Free Full Text]
  3. Chen XQ, Tan I, Ng CH, Hall C, Lim L, and Leung T. Characterization of RhoA-binding kinase ROK{alpha}: implication of the pleckstrin homology domain in ROK{alpha} function using region-specific antibodies. J Biol Chem 277: 12680–12688, 2002.[Abstract/Free Full Text]
  4. Chitaley K and Webb RC. Nitric oxide induces dilation of rat aorta via inhibition of rho-kinase signaling. Hypertension 39: 438–442, 2002.[Abstract/Free Full Text]
  5. Chrissobolis S and Sobey CG. Evidence that rho-kinase activity contributes to cerebral vascular tone in vivo and is enhanced during chronic hypertension: comparison with protein kinase C. Circ Res 88: 774–779, 2001.[Abstract/Free Full Text]
  6. Cocks TM and Angus JA. Endothelium-dependent relaxation of coronary arteries by noradrenaline and serotonin. Nature 305: 627–630, 1983.[CrossRef][Medline]
  7. Davies SP, Reddy H, Caivano M, and Cohen P. Specificity and mechanism of action of some commonly used protein kinase inhibitors. Biochem J 351: 95–105, 2000.[CrossRef][Web of Science][Medline]
  8. Delbosc S, Cristol JP, Descomps B, Mimran A, and Jover B. Simvastatin prevents angiotensin II-induced cardiac alteration and oxidative stress. Hypertension 40: 142–147, 2002.[Abstract/Free Full Text]
  9. Endres M, Laufs U, Huang Z, Nakamura T, Huang P, Moskowitz MA, and Liao JK. Stroke protection by 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase inhibitors mediated by endothelial nitric oxide synthase. Proc Natl Acad Sci USA 95: 8880–8885, 1998.[Abstract/Free Full Text]
  10. Harrison DG. Cellular and molecular mechanisms of endothelial cell dysfunction. J Clin Invest 100: 2153–2157, 1997.[Web of Science][Medline]
  11. Hirata K, Kikuchi A, Sasaki T, Kuroda S, Kaibuchi S, Matsuura Y, Seki H, Saida K, and Takai Y. Involvement of rho p21 in the GTP-enhanced calcium ion sensitivity of smooth muscle contraction. J Biol Chem 267: 8719–8722, 1992.[Abstract/Free Full Text]
  12. Huang PL, Huang Z, Mashimo M, Bloch KD, Moskowitz MA, Bevan JA, and Fishman MC. Hypertension in mice lacking the gene for endothelial nitric oxide synthase. Nature 377: 239–242, 1995.[CrossRef][Medline]
  13. Ishizaki T, Uehata M, Tamechika I, Keel J, Nonomura K, Maekawa M, and Narumiya S. Pharmacological properties of Y-27632, a specific inhibitor of rho-associated kinases. Mol Pharmacol 57: 976–983, 2000.[Abstract/Free Full Text]
  14. Kalinowski L, Dobrucki LW, Brovkovych V, and Malinski T. Increased nitric oxide bioavailability in endothelial cells contributes to the pleiotropic effect of cerivastatin. Circulation 105: 933–938, 2002.[Abstract/Free Full Text]
  15. Kandabashi T, Shimokawa H, Miyata K, Kunihiro I, Kawano Y, Fukata Y, Higo T, Egashira K, Takahashi S, Kaibuchi K, and Takeshita A. Inhibition of myosin phosphatase by upregulated Rho-kinase plays a key role for coronary artery spasm in a porcine model with interleukin-1{beta}. Circulation 101: 1319–1323, 2000.[Abstract/Free Full Text]
  16. Kimura K, Ito M, Amano M, Chihara K, Fukata Y, Nakafuku M, Yamamori B, Feng J, Nakano T, Okawa K, Iwamatsu A, and Kaibuchi K. Regulation of myosin phosphatase by rho and rho-associated kinase (rho-kinase). Science 273: 245–248, 1996.[Abstract]
  17. Kojda G, Laursen JB, Ramasamy S, Kent JD, Kurz S, Burchfield J, Shesely EG, and Harrison DG. Protein expression, vascular reactivity and soluble guanylate cyclase activity in mice lacking the endothelial cell nitric oxide synthase: contributions of NOS isoforms to blood pressure and heart rate control. Cardiovasc Res 42: 206–213, 1999.[Abstract/Free Full Text]
  18. Kureishi Y, Luo Z, Shiojima I, Bialik A, Fulton D, Lefer DJ, Sessa WC, and Walsh K. The HMG-CoA reductase inhibitor simvastatin activates the protein kinase Akt and promotes angiogenesis in normocholesterolemic animals. Nat Med 6: 1004–1010, 2000.[CrossRef][Web of Science][Medline]
  19. Lamping KG and Faraci FM. Role of sex differences and effects of endothelial NO synthase deficiency in responses of carotid arteries to serotonin. Arterioscler Thromb Vasc Biol 21: 523–528, 2001.[Abstract/Free Full Text]
  20. Lamping KG, Marcus ML, and Dole WP. Removal of endothelium potentiates large coronary artery constrictor responses to 5-hydroxytryptamine in vivo. Circ Res 57: 46–54, 1985.[Abstract/Free Full Text]
  21. Laufs U, Endres M, Stagliano N, Amin-Hanjani S, Chui DS, Yang SX, Simoncini T, Yamada M, Rabkin E, Allen PG, Huang PL, Bohm M, Schoen FJ, Moskowitz MA, and Liao JK. Neuroprotection mediated by changes in the endothelial actin cytoskeleton. J Clin Invest 106: 15–24, 2000.[Web of Science][Medline]
  22. Laufs U, Gertz K, Huang P, Nickenig G, Bohm M, Dirnagl U, and Endres M. Atorvastatin upregulates type III nitric oxide synthase in thrombocytes, decreases platelet activation, and protects from cerebral ischemia in normocholesterolemic mice. Stroke 31: 2442–2449, 2000.[Abstract/Free Full Text]
  23. Laufs U, La Fata V, Plutzky J, and Liao JK. Upregulation of endothelial nitric oxide synthase by HMG CoA reductase inhibitors. Circulation 97: 1129–1135, 1998.[Abstract/Free Full Text]
  24. Laufs U and Liao JK. Post-transcriptional regulation of endothelial nitric oxide synthase mRNA stability by Rho GTPase. J Biol Chem 273: 24266–24271, 1998.[Abstract/Free Full Text]
  25. Laufs U and Liao JK. Targeting rho in cardiovascular disease. Circ Res 87: 526–528, 2000.[Free Full Text]
  26. Lee MR, Li L, and Kitazawa T. Cyclic GMP causes Ca2+ desensitization in vascular smooth muscle by activating the myosin light chain phosphatase. J Biol Chem 272: 5063–5068, 1997.[Abstract/Free Full Text]
  27. Masumoto A, Hirooka Y, Shimokawa H, Hironaga K, Setoguchi S, and Takeshita A. Possible involvement of Rho-kinase in the pathogenesis of hypertension in humans. Hypertension 38: 1307–1310, 2001.[Abstract/Free Full Text]
  28. Miyata K, Shimokawa H, Kandabashi T, Higo T, Morishige K, Eto Y, Egashira K, Kaibuchi K, and Takeshita A. Rho-kinase is involved in macrophage-mediated formation of coronary vascular lesions in pigs in vivo. Arterioscler Thromb Vasc Biol 20: 2351–2358, 2000.[Abstract/Free Full Text]
  29. Moncada S, Palmer RMJ, and Higgs EA. Nitric oxide: physiology pathophysiology, and pharmacology. Pharmacol Rev 43: 109–142, 1991.[Web of Science][Medline]
  30. Sakurada S, Okamoto H, Takuwa N, Sugimoto N, and Takuwa Y. Rho activation in excitatory agonist-stimulated vascular smooth muscle. Am J Physiol Cell Physiol 281: C571–C578, 2001.[Abstract/Free Full Text]
  31. Sato M, Tani E, Fujikawa H, and Kaibuchi K. Involvement of rho-kinase-mediated phosphorylation of myosin light chain in enhancement of cerebral vasospasm. Circ Res 87: 195–200, 2000.[Abstract/Free Full Text]
  32. Sauzeau V, Le Jeune H, Cario-Toumaniantz C, Smolenski A, Lohmann SM, Bertoglio J, Chardin P, Pacaud P, and Loirand G. Cyclic GMP-dependent protein kinase signaling pathway inhibits RhoA-induced Ca2+ sensitization of contraction in vascular smooth muscle. J Biol Chem 275: 21722–21729, 2000.[Abstract/Free Full Text]
  33. Seasholtz TM, Zhang T, Morissette MR, Howes AL, Yang AH, and Brown JH. Increased expression and activity of RhoA are associated with increased DNA synthesis and reduced p27(Kip1) expression in the vasculature of hypertensive rats. Circ Res 89: 488–495, 2001.[Abstract/Free Full Text]
  34. Shimokawa H. Rho-kinase as a novel therapeutic target in treatment of cardiovascular diseases. J Cardiovasc Pharmacol 39: 319–327, 2002.[CrossRef][Web of Science][Medline]
  35. Somlyo AP and Somlyo AV. Signal transduction by G-proteins, rho-kinase and protein phosphatase to smooth muscle and non-muscle myosin II. J Physiol 522: 177–185, 2000.[Abstract/Free Full Text]
  36. Takemoto M and Liao JK. Pleiotropic effects of 3-hydroxy-3-methylglutaryl coenzyme a reductase inhibitors. Arterioscler Thromb Vasc Biol 21: 1712–1719, 2001.[Abstract/Free Full Text]
  37. Uehata M, Ishizaki T, Satoh H, Ono T, Kawahara T, Morishita T, Tamakawa H, Yamagami K, Inui J, Maekawa M, and Narumiya S. Calcium sensitization of smooth muscle mediated by a Rho-associated protein kinase in hypertension. Nature 389: 990–994, 1997.[CrossRef][Medline]
  38. Wagner AH, Schwabe O, and Hecker M. Atorvastatin inhibition of cytokine-inducible nitric oxide synthase expression in native endothelial cells in situ. Br J Pharmacol 136: 143–149, 2002.[CrossRef][Web of Science][Medline]
  39. Waldron GJ, Ding H, Lovren F, Kubes P, and Triggle CR. Acetylcholine-induced relaxation of peripheral arteries isolated from mice lacking endothelial nitric oxide synthase. Br J Pharmacol 128: 653–658, 1999.[CrossRef][Web of Science][Medline]
  40. Wu X, Somlyo AV, and Somlyo AP. Cyclic GMP-dependent stimulation reverses G-protein-coupled inhibition of smooth muscle myosin light chain phosphatase. Biochem Biophys Res Commun 220: 658–663, 1996.[CrossRef][Web of Science][Medline]
  41. Yamakawa T, Tanaka S, Numaguchi K, Yamakawa Y, Motley ED, Ichihara S, and Inagami T. Involvement of Rho-kinase in angiotensin II-induced hypertrophy of rat vascular smooth muscle cells. Hypertension 35: 313–318, 2000.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 CirculationHome page
M. F. Kircher, J. Grimm, F. K. Swirski, P. Libby, R. E. Gerszten, J. R. Allport, and R. Weissleder
Noninvasive In Vivo Imaging of Monocyte Trafficking to Atherosclerotic Lesions
Circulation, January 22, 2008; 117(3): 388 - 395.
[Abstract] [Full Text] [PDF]

Home page
 StrokeHome page
S. Chrissobolis and C. G. Sobey
Recent Evidence for an Involvement of Rho-Kinase in Cerebral Vascular Disease
Stroke, August 1, 2006; 37(8): 2174 - 2180.
[Abstract] [Full Text] [PDF]

Home page
 J. Pharmacol. Exp. Ther.Home page
K. Budzyn, P. D. Marley, and C. G. Sobey
Opposing Roles of Endothelial and Smooth Muscle Phosphatidylinositol 3-Kinase in Vasoconstriction: Effects of Rho-Kinase and Hypertension
J. Pharmacol. Exp. Ther., June 1, 2005; 313(3): 1248 - 1253.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
287/2/R342    most recent
00156.2004v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (13)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Budzyn, K.
Right arrow Articles by Sobey, C. G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Budzyn, K.
Right arrow Articles by Sobey, C. G.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2004 by the American Physiological Society.