Skip Navigation

Cardiovascular Research 2006 72(3):456-463; doi:10.1016/j.cardiores.2006.09.007
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow E-letters: Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when E-letters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Song, J.
Right arrow Articles by Martin, D. S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Song, J.
Right arrow Articles by Martin, D. S.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Copyright © 2006, European Society of Cardiology

Androgens potentiate renal vascular responses to angiotensin II via amplification of the Rho kinase signaling pathway

Jin Song, Curtis K. Kost, Jr. and Douglas S. Martin*

Basic Biomedical Sciences, Sanford School of Medicine, University of South Dakota, 414 East Clark Street, Vermillion, SD 57069, United States

* Corresponding author. Tel.: +1 605 677 5162; fax: +1 605 677 6381. Email address: doug.martin{at}usd.edu

Received 31 July 2006; revised 11 September 2006; accepted 13 September 2006


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objectives: This study assessed whether the Rho kinase signaling pathway contributes to androgenic amplification of angiotensin II (Ang II) induced pressor and renal constrictor responses.

Methods: Mean arterial pressure (MAP) responses to angiotensin II receptor 1 (AT1) inhibition were measured in conscious male New Zealand genetically hypertensive rats (NZGH) subjected to sham operation, castration or castration+testosterone replacement. MAP and renal vascular resistance (RVR) responses to Ang II were recorded with and without a Rho kinase inhibitor, fasudil, in anesthetized NZGH. Western blot was used to analyze target protein expression in the kidney.

Results: MAP responses to AT1 receptor inhibition and exogenous Ang II were attenuated in castrated NZGH. The increase in RVR (mm Hg/ml/min/g kidney) at the maximum dose of Ang II was significantly lower in castrated NZGH than in sham operated NZGH. Testosterone replacement restored RVR responses to Ang II in castrated rats. Fasudil treatment reduced both MAP and RVR responses to Ang II in each group. In addition, the differential MAP and RVR responses to Ang II amongst the three groups were significantly attenuated by Rho kinase inhibition. Western blot showed that Rho kinase protein expression was reduced by castration, while testosterone replacement restored the Rho kinase protein levels in castrated rats. The phosphorylation of myosin phosphatase target subunit 1 (MYPT1), a downstream target of Rho kinase, was also increased by androgens.

Conclusions: Collectively, these results indicate that androgens potentiate Ang II-induced renal vascular responses, an effect mediated at least partly via up-regulation of the Rho kinase signaling pathway.

KEYWORDS Gender; Angiotensin; Protein kinase; Vasoconstriction; Hypertension


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
A gender-related difference in the regulation of blood pressure has been documented in both human and animal models [1–4]. Recent studies showed that males are at greater risk for development of hypertension than age-matched premenopausal females. The factors mediating these sex based differences are complex but androgens appear to be important at least under certain conditions [3,5].

Accumulating evidence suggests that androgens influence hypertension development and its sequelae via an interaction with the kidney. Early studies by Ganten et al. showed that surgical or chemical castration of the spontaneously hypertensive rat (SHR) resulted in a marked reduction in hypertension development [6]. Similarly a subsequent series of elegant experiments by Reckelhoff and colleagues showed that testosterone was essential for the full development of hypertension in the SHR [7]. Androgens also contribute to hypertension-induced renal injury [8,9]. In our laboratory we observed a similar pro-hypertensive effect of testosterone in another genetic model of hypertension, the New Zealand genetically hypertensive rat (NZGH) [10]. Thus considerable data suggest that androgens exacerbate the development of high blood pressure. However the mechanisms by which androgens amplify the development of hypertension remain to be fully defined.

The kidneys are critical in regulating sodium and water balance and blood pressure. Moreover, hypertension is closely related to renal dysfunction [11,12]. Ashton reported that male NZGH exhibit altered sodium and water handling compared with female NZGH which may contribute to the sexual dimorphism in hypertension in this rat model [13]. The mechanisms underlying the renal effect of androgens are not fully elucidated. One possibility is that androgens may affect renal vascular function. We also found that androgens modulated the renal vascular responses to angiotensin II (Ang II) in NZGH [10]. Renal vascular resistance (RVR) responses to Ang II infusion were significantly lower in castrated NZGH than sham operated NZGH and castrated NZGH with testosterone replacement [10].

Activation of Rho A/Rho kinase is one of the major signaling pathways involved in Ang II-induced vasoconstriction [14,15]. Ang II binding to Ang II receptor 1 (AT1) causes activation of the monomeric small G protein Rho A and its target, Rho kinase [16]. Activated Rho kinase inhibits myosin light chain (MLC) phosphatase, maintaining MLC in the phosphorylated state, therefore amplifying vasoconstriction [17]. Convincing evidence has shown that the Rho kinase signaling pathway is involved in hypertension development and that Rho kinase activity and expression are enhanced in hypertensive blood vessels [18,19]. Sex steroids may modulate Rho kinase effects on smooth muscle contractile function. Estrogen or progesterone treatment of ovariectomized rats increased expression of Rnd1, which acted as a brake on the RhoA/Rho kinase pathway and reduced calcium sensitivity in smooth muscle [20]. Similarly, Chrissobolis et al. found that estrogen suppresses Rho kinase function in the cerebral circulation in vivo [21]. However, whether androgens potentiate renal vascular reactivity to Ang II via an effect on the Rho kinase pathway in NZGH model is still not clear.

Accordingly, this study was designed to further investigate the mechanisms of androgen amplification of Ang II-induced renal vasoconstriction. We hypothesize that androgens potentiate renal vascular responses to Ang II, at least in part, via up-regulation of Rho kinase signaling pathway in NZGH. This hypothesis was tested by assessing pressor and renal vascular reactivity to Ang II before and after impairment of Rho kinase function. In addition, Western blot approaches were used to assess expression and activity of Rho kinase.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1. Rats
Male NZGH (Charles River Laboratory, Wilmington, MA) were obtained at 4 weeks of age and maintained on commercially available phytoestrogen-free rat chow (Harland Teklad 2016) and tap water in an environment with a 12-h/12-h light/dark cycle. All experiments and protocols were performed in accordance with the regulations set forth by the National Institutes of Health Council on Animal Care and were approved by the Institutional Animal Care and Use Committee at the University of South Dakota.

2.2. Castration and chronic testosterone treatment
At 5 weeks of age, rats were subjected to sham operation (Sham) or castration (Cas) as described previously [10]. Following the castration procedure, one group of castrated rats was provided testosterone replacement (Cas+T) (testosterone pellet 100 mg, 90 day release, Innovative Research of America), which was previously shown to produce plasma testosterone concentrations similar to those of age-matched sham operated NZGH [10].

2.3. In vivo experimental protocol
2.3.1. Conscious rats
At 16–17 weeks of age, one cohort of NZGH was anesthetized with isoflurane, and catheters were implanted in the femoral artery for measuring mean arterial blood pressure (MAP) and in the femoral vein for injection of an AT1 receptor antagonist, L-158809 (Merck Research Laboratories, PA). After 48 h of recovery, MAP was measured when the animals were calm. Baseline MAP was recorded in the first 30-min. Then the rats were given bolus injections of L-158809 (1–1000 µg/kg in 0.3 ml saline, IV) and MAP was recorded for 20-min after each dose of L-158809 to construct dose–MAP response curves to AT1 inhibition.

2.3.2. Anesthetized rats
A separate cohort of rats was anesthetized with an injection of Inactin (100 mg/kg, IP) and placed on a temperature-regulated surgical table to maintain body temperature at 37 °C. A tracheotomy was performed to facilitate breathing. Catheters were placed in the femoral artery for measuring MAP and in the femoral vein for infusion of Ang II. Another catheter was inserted into the right jugular vein for infusion of saline or saline with fasudil, a Rho kinase inhibitor, (Tocris, MO) at 3 ml/h. The abdomen was opened and an ultrasonic flow probe (Transonic) was placed on the left renal artery to measure the renal blood flow (RBF). After 60-min of stabilization, the rats were given a bolus injection of enalapril, an angiotensin-converting enzyme (ACE) inhibitor, (1 mg/kg in 0.3 ml saline, IV) to inhibit endogenous Ang II production. Then MAP and RBF were measured before and during intravenous Ang II infusion (20, 40 and 80 ng/kg/min). Each infusion lasted for 30 min. After recovery from the dose response curves, the rats were treated with fasudil (1 mg/kg bolus injection, followed by 33 µg/kg/min infusion, IV). After 20 min of stabilization, MAP and RBF responses to Ang II were repeated during the fasudil infusion. Renal vascular resistance (RVR) was calculated as MAP divided by RBF. At the end of the experiment, the rats were euthanized and the kidneys were removed and weighed.

2.4. Western blot analysis
A separate cohort of NZGH was euthanized and the kidneys were harvested and fast frozen. Western blot analysis was performed on renal cortex punches. The tissue samples were homogenized in RIPA buffer (50 mM Tris pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% Deoxycholic acid, 0.1% SDS, 2 mM EDTA, 10 mM NaF, 10 µg/ml leupeptin, 10 µg/ml pepstatin, and 20 µg/ml aprotinin). Equal amounts of protein (50 µg/well) were loaded onto each lane of 7% acrylamide gels, which were then subjected to SDS-PAGE electrophoresis and transferred to PVDF membranes (Bio-Rad). Protein expression of Rho kinase (ROCK II) was analyzed by Western blot with rabbit polyclonal anti-ROCK II antibody (1:1000, Upstate, NY). Actin served as a loading control. Data were expressed as the ratio of ROCK II to actin.

Rho kinase phosphorylates the myosin phosphatase target subunit 1 (MYPT1) of myosin light chain phosphatase at Thr-696, leading to the inhibition of its activity [22,23]. Accordingly, the ratio of phosphorylated MYPT1 to total MYPT1 was used as an index of the Rho kinase activity [24]. Total MYPT1 expression was determined by Western blot of renal cortex punches using a rabbit polyclonal anti-MYPT1 antibody (1:1000, Upstate, NY). Phosphorylated MYPT1 was detected using a rabbit polyclonal anti-phospho-MYPT1 Thr-696 antibody (1:1000, Upstate, NY). The protein bands were visualized using fluorescently-labeled secondary antibodies and subsequent image detection (Odyssey Imager, LiCor Biosciences), then quantified by densitometric analysis software on the Odyssey Imager.

2.5. Statistical analysis
All values are presented as the mean±SEM. One way analysis of variance (ANOVA) was used to analyze baseline values in conscious rats and Western blot analysis. Two-Factor ANOVA for repeated measures was used to analyze baseline values in anesthetized rats, and dose responses to L-158809 and Ang II (grouping factors: surgery x drug). Post hoc comparisons were performed with Student Newman Keuls test (Prism 4, Graph Pad Software, San Diego, CA). Statistical significance was accepted at P<0.05.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1. Conscious rats: baseline values and depressor responses to AT1 inhibition
Castration significantly reduced baseline MAP values in conscious NZGH (Sham 192±2 mm Hg vs. Cas 162±4 mm Hg), whereas testosterone replacement in castrated rats restored MAP (189±3 mm Hg). As shown in Fig. 1, bolus injections of the AT1 blocker, L-158809, caused significant dose-dependent decreases of MAP in each group of conscious NZGH rats. However the decrement of MAP was significantly attenuated in the castrated group compared to sham and castration with testosterone groups. At the highest dose of L-158809, these decreases in MAP were as follows: Sham 33±2 mm Hg vs. Cas 23±3 mm Hg vs. Cas+T 31±5 mm Hg.


Figure 1
View larger version (21K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 1 Depressor responses to administration of an AT1 receptor antagonist, L-158809, (1–1000 µg/kg) in conscious sham operated (Sham N=5), castrated (Cas N=5) and castrated+testosterone replacement (Cas+T N=5) NZGH at 16–17 weeks of age. Values are mean±SEM. *P<0.05 Sham vs. Cas. #P<0.05 Cas+T vs. Cas.

 
3.2. Pressor and renal vascular responses to Ang II in anesthetized rats
3.2.1. Effect of androgen manipulation
The baseline MAP and RVR values after endogenous Ang II inhibition by enalapril in anesthetized rats are shown in Table 1. There were no significant differences in baseline values amongst different surgery groups. However, compared to the baseline values, MAP was decreased during fasudil infusion.


View this table:
[in this window]
[in a new window]

  		Table 1 Baseline values

 
As shown in Fig. 2A, cumulative infusions of Ang II (20, 40, and 80 ng/kg/min) were associated with dose-dependent increases of MAP in each group. The range of MAP responses to Ang II in sham operated NZGH was between approximately 48±5 and 74±7 mm Hg. Castration significantly attenuated MAP responses to Ang II. MAP responses to Ang II ranged between 33±3 and 52±2 mm Hg in castrated NZGH. Testosterone replacement of castrated NZGH significantly increased Ang II-induced MAP responses such that these responses were similar to those recorded in sham operated rats. Thus testosterone replacement of castrated NZGH restored the pressor responses to Ang II.


Figure 2
View larger version (26K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 2 Mean arterial blood pressure (MAP) responses to Ang II (20, 40 and 80 ng/kg/min) in anesthetized sham operated (Sham N=5), castrated (Cas N=5) and castrated+testosterone replacement (Cas+T N=5) NZGH at 16–17 weeks of age. (A) MAP responses in the absence of fasudil. (B) MAP responses in the presence of fasudil. Values are mean±SEM. *P<0.05 and **P<0.01 Sham vs. Cas. ##P<0.01 Cas+T vs. Cas.

 
The renal vascular responses to Ang II (20, 40, and 80 ng/kg/min) are shown in Fig. 3. There was a dose-dependent increase of RVR in response to Ang II infusion in each group (Fig. 3A). However, RVR responses to Ang II were significantly attenuated in male NZGH subjected to castration. Testosterone replacement of castrated NZGH restored RVR responses to Ang II.


Figure 3
View larger version (31K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 3 Renal vascular resistance (RVR) responses to Ang II (20, 40 and 80 ng/kg/min) in anesthetized sham operated (Sham N=5), castrated (Cas N=5) and castrated+testosterone replacement (Cas+T N=5) NZGH at 16–17 weeks of age. (A) RVR responses in the absence of fasudil. (B) RVR responses in the presence of fasudil. Values are mean±SEM. *P<0.05 and **P<0.01 Sham vs. Cas. #P<0.05 and ##P<0.01 Cas+T vs. Cas.

 
3.2.2. Effect of fasudil treatment
Fasudil infusion significantly attenuated pressor responses to Ang II in each group as shown in Fig. 2B. Moreover the inhibitory effect of fasudil was significantly greater in both sham operated NZGH and castrated NZGH with testosterone replacement compared to the castrated rats (Fig. 4A). Accordingly, the differences amongst the androgen replete and castrated NZGH were greatly attenuated during fasudil treatment (Fig. 2B).


Figure 4
View larger version (19K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 4 This graph depicts the fasudil inhibited component of Ang II-induced pressor (A) and RVR (B) responses. These values were obtained by calculating the difference between the MAP and RVR responses achieved at the highest dose of Ang II before fasudil treatment from the corresponding responses obtained after fasudil treatment in sham operated (Sham N=5), castrated (Cas N=5) and castrated+testosterone replacement (Cas+T N=5) NZGH at 16–17 weeks of age. Values are mean±SEM. *P<0.05 and **P<0.01 Sham vs. Cas. #P<0.05 Cas+T vs. Cas.

 
A similar pattern was observed for the renal vascular resistance responses. Fasudil treatment significantly attenuated renal constrictor responses to Ang II (Fig. 3B). The fasudil-induced attenuation of Ang II renal vasoconstriction was greater in the androgen replete NZGH compared to the castrated NZGH (Fig. 4B). Although there remained a significant difference in the renal vascular resistance response to Ang II at the highest dose, fasudil treatment diminished the differences in renal vascular responses amongst NZGH with and without testosterone (Fig. 3B). Thus fasudil treatment was effective in greatly suppressing the differential Ang II-induced pressor and renal vascular resistance responses observed between androgen replete and androgen depleted NZGH.

3.2.3. Effect of androgens on Rho kinase expression
In order to determine whether the functional changes in Ang II-induced dose responses were due to any change of Rho kinase protein expression level, the protein abundance of Rho kinase was analyzed by Western blot. As shown in Fig. 5, Rho kinase (ROCK II) signal (~150 kDa) was detected in the renal cortex punches of each of the experimental groups. Quantification of the data showed that there was a significant decrease (~25%) of ROCK II protein expression in castrated NZGH compared to sham operated rats. Testosterone replacement significantly increased the protein level of ROCK II in castrated rats.


Figure 5
View larger version (25K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 5 Rho kinase (ROCK II) protein expression as assessed by Western blot in renal cortex obtained from sham operated (Sham N=8), castrated (Cas N=8) and castrated+testosterone replacement (Cas+T N=8) NZGH at 16–17 weeks of age. The top panel shows an example of ROCK II bands (~150 kDa) and actin bands (~42 kDa). The lower panel shows the quantification of the blots expressed as the ROCK II/actin ratio. Values are mean±SEM. *P<0.05 Sham vs. Cas. ##P<0.01 Cas+T vs. Cas. &P<0.05 Cas+T vs. Sham.

 
3.2.4. Effect of androgens on Rho kinase activity
Rho kinase inhibits the activity of myosin light chain phosphatase by phosphorylation of the MYPT1 subunit on Thr-696 [23]. Therefore, the ratio of phosphorylated MYPT1 to total MYPT1 was used as an index of Rho kinase activity. The top panel of Fig. 6 shows an example of both phosphorylated and total MYPT1 bands. The summary data illustrated in the bottom panel show that the relative phosphorylation of MYPT1 was significantly lower in castrated rats than sham operated and castrated rats with testosterone treatment.


Figure 6
View larger version (28K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 6 Relative phosphorylation level of MYPT1 in the homogenates of renal cortex tissue obtained from sham operated (Sham N=8), castrated (Cas N=8) and castrated+testosterone replacement (Cas+T N=8) NZGH at 16–17 weeks of age. The top panel shows an example of phosphorylated (P-MYPT1) and total MYPT1 (T-MYPT1) bands (~130 kDa). The lower panel shows the quantification of the bands as the P-MYPT1/T-MYPT1 ratio. *P<0.05 Sham vs. Cas. #P<0.05 Cas+T vs. Cas.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
In the present study, we tested the hypothesis that Rho kinase plays a role in androgen modulation of renal vascular responses to Ang II. Our data demonstrate the following: 1) Depressor responses to AT1 receptor blockade were exaggerated in androgen replete NZGH compared to castrated NZGH. 2) There were significantly greater pressor and renal vasoconstrictor responses to exogenous Ang II in sham operated NZGH and castrated NZGH with testosterone replacement compared to castrated NZGH. 3) Differences in Ang II dose responses amongst these groups were markedly attenuated by fasudil treatment. 4) Both protein expression and activity of Rho kinase in the kidney were reduced by castration, whereas testosterone replacement reversed the effect of castration. Together, these findings are consistent with the view that androgens potentiate renal vascular responses to Ang II, at least in part, via the amplification of Rho kinase signaling in NZGH.

Gender-associated differences in blood pressure regulation have been demonstrated in both human and animal studies [1,3]. Although the exact mechanisms are not clear, several observations documented that androgens have an important role in blood pressure regulation [1,2,5,6]. A study by Ganten et al. indicated that castration of male SHR at 3 weeks of age significantly decreased systolic blood pressure recorded at 25 weeks of age by approximately 40 mm Hg [6]. Similarly, Reckelhoff, and Chen and Meng also reported that castration at an early age reduced blood pressure observed in adult male SHR [2,5]. In addition, administration of testosterone reversed the antihypertensive effects of castration and androgen receptor antagonist treatment mimicked the effects of castration, suggesting that sexual dimorphism of blood pressure in SHR is androgen-dependent [5,25]. In the current studies, we showed that MAP was reduced by approximately 30 mm Hg in castrated conscious NZGH, another animal model of spontaneous hypertension [26,27].

It is well accepted that renal mechanisms play an important role in sodium and water regulation and long-term blood pressure control [11,12,28]. Renal dysfunction has been documented in hypertension in both humans and animal models [11]. Despite considerable interest in gender-associated differences in high blood pressure, relatively few studies have addressed the impact of androgens on the renal function [1]. There are multiple ways by which androgens may affect renal blood pressure control mechanisms. One possibility is that androgens may affect renal vascular reactivity. Evidence from the SHR model suggests that hypertension is due, in part, to enhanced effects of Ang II on the renal vasculature [29]. In addition, accumulating evidence indicates that androgens may up-regulate the renin–angiotensin system, which contributes to the pro-hypertensive effect of androgens [30]. Consistent with this finding, we observed that blood pressure responses to AT1 inhibition were augmented in androgen replete groups compared to castrated NZGH, suggesting a modulatory effect of androgens on systemic responses to endogenous Ang II. In order to determine whether androgens modulate Ang II-induced renal vasoconstriction, we measured MAP and RVR in response to exogenous Ang II infusion in anesthetized NZGH in vivo. Our data showed that MAP responses to exogenous Ang II infusion were attenuated in castrated NZGH and were restored by testosterone treatment. These data suggest that androgens amplify the overall Ang II-induced systemic vascular tone. Furthermore, RVR responses to Ang II were attenuated by castration, while testosterone replacement restored these responses in castrated rats confirming our earlier work [10]. Since enhanced renal constriction responses to Ang II have been linked to hypertension, these observations are consistent with the possibility that androgens amplify the development of hypertension, at least in part, by increasing the renal vasoconstrictor response to Ang II.

The vasoconstriction induced by Ang II involves the AT1 receptor and its downstream signaling pathways [15]. Therefore possible mechanisms by which androgens may affect Ang II-induced renal vasoconstriction include up-regulation of the AT1 receptor or its downstream signaling pathways. Although it was reported that androgens increased AT1 receptor expression in male reproductive tissue [31], our previous studies found that neither mRNA nor protein expression of AT1 receptor in the kidney was significantly changed by castration or testosterone treatment [10]. Thus the functional differences in response to Ang II infusion amongst androgen replete and castrated NZGH may be not related to receptor expression but rather to androgen modulation of the postreceptor signaling pathways coupled to the AT1 receptor. It is well accepted that classic vascular smooth muscle contraction involves Ca2+-dependent mechanisms [17,32]. During vascular smooth muscle activation, intracellular Ca2+ is increased and the Ca2+–calmodulin complex activates myosin light chain (MLC) kinase, which phosphorylates MLC and thereby leads to vasoconstriction [24]. In addition to classic Ca2+-dependent mechanisms, Ca2+ sensitization mechanisms also contribute to vascular smooth muscle contraction [33]. Besides MLC kinase, phosphorylation of MLC is also regulated by MLC phosphatase, which can dephosphorylate and inhibit MLC, resulting in vasodilation [33]. Upon binding to the AT1 receptor, Ang II triggers the activation of the small G protein Rho A and its downstream target, Rho kinase [15]. Activated Rho kinase phosphorylates a regulatory subunit (MYPT1) of MLC phosphatase at Thr-696, inhibiting its activity [33]. Thereby more MLC is maintained in the phosphorylated form leading to augmented vasoconstriction independent of changes in intracellular Ca2+ concentrations. Rho kinase activation appears to contribute importantly to Ang II-induced vasoconstriction. Ang II mediated cerebral vasoconstriction in mice was prevented by a Rho kinase inhibitor, Y-27632 [34]. Cavarape et al. demonstrated that Rho kinase inhibition caused a vasodilator effect in both preglomerular and postglomerular vessels, with a significant increase in glomerular blood flow suggesting that Rho kinase plays an important role in the control of renal vasoconstriction [35,36]. Our findings are consistent with this view. Fasudil infusion markedly attenuated the renal vascular responses to Ang II in sham operated, castrated and castrated+testosterone replacement NZGH, providing further support for the involvement of Rho kinase in Ang II-induced renal vasoconstriction.

Convincing evidence from both animal and human models indicates that Rho kinase mediated Ca2+ sensitization plays an important role in the pathogenesis of hypertension [19,37]. Increased Rho A/Rho kinase signaling contributes to high peripheral vascular resistance in hypertension [18]. Both expression and activity of Rho A/Rho kinase are increased in carotid and mesentery blood vessels in hypertensive animals [18]. The Rho kinase inhibitor, Y-27632, reduced spontaneous tone in aortic rings from Ang II-induced hypertensive rats [38]. Moreover, enhanced activation of Rho A by Ang II has also been found in preglomerular microvascular smooth muscle cells derived from the kidney of SHR [39]. Despite compelling evidence implicating the Rho kinase pathway in hypertension, few studies have explored the possible interactions between sex hormones and the Rho kinase pathway in vascular smooth muscle. Chrissobolis et al. reported that Rho kinase function in the cerebral circulation is suppressed in female compared with male Sprague–Dawley rats in vivo [21], while Loirand et al. showed that estrogen treatment increased the expression of Rnd1, an integral inhibitory component of the Rho kinase pathway, in the aorta [20]. Our current data suggest that androgens also modulate the Rho kinase signaling pathway in the systemic and renal vasculature. Differences in MAP and RVR responsiveness to Ang II amongst the castrated and androgen replete NZGH were markedly attenuated by fasudil treatment suggesting that androgens may amplify Ang II signaling via the Rho Kinase pathway. Compatible with this possibility, Western blot analysis found that the protein expression of Rho kinase in kidney cortex tissue was attenuated by castration, while chronic testosterone replacement enhanced Rho kinase expression significantly. In contrast to our findings in the kidney, castration was shown to increase Rho kinase protein levels in penile cavernosal tissue [40]. Thus there appears to be regional specificity in the effects of androgens on the vascular Rho kinase pathway. We also observed that the relative phosphorylation of MYPT1 was decreased in the kidney of castrated rats compared to androgen replete rats, suggestive of a castration-induced reduction in Rho kinase activity. Together, both functional and cellular data obtained in this study suggest that Rho kinase contributes to androgenic amplification of renal vascular responses to Ang II.

4.1. Perspectives
Amplification of vascular responses is a common denominator in many forms of hypertension. A series of studies by Reckelhoff and Granger demonstrated that androgens modulate renal function in SHR [8]. Aging male SHR showed increased proteinuria, renal vascular resistance and glomerular injury, and decreased glomerular filtration, while these pathologic changes can be attenuated by castration, suggesting important effects of androgens on the kidney [41]. Our data suggested that androgens amplify the renal vasoconstrictor effects of Ang II via modulation of the Rho kinase signaling pathway which may contribute to hypertension-induced renal pathology. Indeed, recent data indicate that Rho kinase inhibition exerted reno-protective effects in hypertensive rats [42–45]. Moreover, preliminary work in humans showed that Rho kinase inhibition exerts a significant antihypertensive effect [46], indicating the clinical relevance of this pathway. Thus, the Rho kinase signaling pathway is an attractive target for further development of antihypertensive and renal protective agents.


   Acknowledgements
 
This work was supported by grants #HL063053 (DSM) and #HL074852 (CKK) from the National Heart Lung and Blood Institute of the NIH and #0515443Z from the American Heart Association (JS).


   Notes
 
Time for primary review 24 days


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

  1. Dubey R.K., Oparil S., Imthurn B., Jackson E.K. Sex hormones and hypertension. Cardiovasc Res (2002) 53:688–708.[Abstract/Free Full Text]
  2. Reckelhoff J.F. Gender differences in the regulation of blood pressure. Hypertension (2001) 37:1199–1208.[Abstract/Free Full Text]
  3. Reckelhoff J.F. Sex steroids, cardiovascular disease, and hypertension: unanswered questions and some speculations. Hypertension (2005) 45:170–174.[Free Full Text]
  4. Rowland N.E., Fregly M.J. Role of gonadal hormones in hypertension in the Dahl salt-sensitive rat. Clin Exp Hypertens A (1992) 14:367–375.[Web of Science][Medline]
  5. Chen Y.F., Meng Q.C. Sexual dimorphism of blood pressure in spontaneously hypertensive rats is androgen dependent. Life Sci (1991) 48:85–96.[CrossRef][Web of Science][Medline]
  6. Ganten U., Schroder G., Witt M., Zimmermann F., Stock D., Stock G. Sexual dimorphism of blood pressure in spontaneously hypertensive rats: effects of anti-androgen treatment. J Hypertens (1989) 7:721–726.[Web of Science][Medline]
  7. Reckelhoff J.F., Zhang H., Granger J.P. Testosterone exacerbates hypertension and reduces pressure-natriuresis in male spontaneously hypertensive rats. Hypertension (1998) 31:435–439.[Abstract/Free Full Text]
  8. Reckelhoff J.F., Granger J.P. Role of androgens in mediating hypertension and renal injury. Clin Exp Pharmacol Physiol (1999) 26:127–131.[CrossRef][Web of Science][Medline]
  9. Ji H., Menini S., Mok K., Zheng W., Pesce C., Kim J., et al. Gonadal steroid regulation of renal injury in renal wrap hypertension. Am J Physiol Renal Physiol (2005) 288:F513–F520.[Abstract/Free Full Text]
  10. Song J., Kost C.K. Jr., Martin D.S. Androgens augment renal vascular responses to ANG II in New Zealand genetically hypertensive rats. Am J Physiol Regul Integr Comp Physiol (2006) 290:R1608–R1615.[Abstract/Free Full Text]
  11. Hall J.E. The kidney, hypertension, and obesity. Hypertension (2003) 41:625–633.[Abstract/Free Full Text]
  12. Grisk O., Rettig R. Renal transplantation studies in genetic hypertension. News Physiol Sci (2001) 16:262–265.[Abstract/Free Full Text]
  13. Ashton N., Balment R.J. Sexual dimorphism in renal function and hormonal status of New Zealand genetically hypertensive rats. Acta Endocrinol (Copenh) (1991) 124:91–97.[Abstract/Free Full Text]
  14. Touyz R.M., Berry C. Recent advances in angiotensin II signaling. Braz J Med Biol Res (2002) 35:1001–1015.[Web of Science][Medline]
  15. Touyz R.M. The role of angiotensin II in regulating vascular structural and functional changes in hypertension. Curr Hypertens Rep (2003) 5:155–164.[Web of Science][Medline]
  16. Cavarape A., Bauer J., Bartoli E., Endlich K., Parekh N. Effects of angiotensin II, arginine vasopressin and thromboxane A2 in renal vascular bed: role of Rho-kinase. Nephrol Dial Transplant (2003) 18:1764–1769.[Abstract/Free Full Text]
  17. Somlyo A.P., Somlyo A.V. Signal transduction by G-proteins, Rho-kinase and protein phosphatase to smooth muscle and non-muscle myosin II. J Physiol (2000) 522(Pt 2):177–185.[Abstract/Free Full Text]
  18. Mukai Y., Shimokawa H., Matoba T., Kandabashi T., Satoh S., Hiroki J., et al. Involvement of Rho-kinase in hypertensive vascular disease: a novel therapeutic target in hypertension. FASEB J (2001) 15:1062–1064.[Free Full Text]
  19. Masumoto A., Hirooka Y., Shimokawa H., Hironaga K., Setoguchi S., Takeshita A. Possible involvement of Rho-kinase in the pathogenesis of hypertension in humans. Hypertension (2001) 38:1307–1310.[Abstract/Free Full Text]
  20. Loirand G., Cario-Toumaniantz C., Chardin P., Pacaud P. The Rho-related protein Rnd1 inhibits Ca2+ sensitization of rat smooth muscle. J Physiol (1999) 516(Pt 3):825–834.[Abstract/Free Full Text]
  21. Chrissobolis S., Budzyn K., Marley P.D., Sobey C.G. Evidence that estrogen suppresses Rho-kinase function in the cerebral circulation in vivo. Stroke (2004) 35:2200–2205.[Abstract/Free Full Text]
  22. Guilluy C., Sauzeau V., Rolli-Derkinderen M., Guerin P., Sagan C., Pacaud P., et al. Inhibition of RhoA/Rho kinase pathway is involved in the beneficial effect of sildenafil on pulmonary hypertension. Br J Pharmacol (2005) 146:1010–1018.[CrossRef][Web of Science][Medline]
  23. Moriki N., Ito M., Seko T., Kureishi Y., Okamoto R., Nakakuki T., et al. RhoA activation in vascular smooth muscle cells from stroke-prone spontaneously hypertensive rats. Hypertens Res (2004) 27:263–270.[CrossRef][Web of Science][Medline]
  24. Lee D.L., Webb R.C., Jin L. Hypertension and RhoA/Rho-kinase signaling in the vasculature: highlights from the recent literature. Hypertension (2004) 44:796–799.[Abstract/Free Full Text]
  25. Reckelhoff J.F., Zhang H., Srivastava K., Granger J.P. Gender differences in hypertension in spontaneously hypertensive rats: role of androgens and androgen receptor. Hypertension (1999) 34:920–923.[Abstract/Free Full Text]
  26. Simpson F.O., Phelan E.L., Clark D.W., Jones D.R., Gresson C.R., Lee D.R., et al. Studies on the New Zealand strain of genetically hypertensive rats. Clin Sci Mol Med Suppl (1973) 45(Suppl_1):15s–21s.[Medline]
  27. Simpson F.O., Ledingham J.M., Phelan E.L. Body sodium level in hypertensive and normotensive rats: effects of dietary sodium levels and sodium loads. J Cardiovasc Pharmacol (1984) 6(Suppl 1):S121–S125.[CrossRef][Web of Science][Medline]
  28. Hall J.E., Mizelle H.L., Hildebrandt D.A., Brands M.W. Abnormal pressure natriuresis. A cause or a consequence of hypertension? Hypertension (1990) 15:547–559.[Abstract/Free Full Text]
  29. Kost C.K. Jr., Herzer W.A., Li P., Jackson E.K. Vascular reactivity to angiotensin II is selectively enhanced in the kidneys of spontaneously hypertensive rats. J Pharmacol Exp Ther (1994) 269:82–88.[Abstract/Free Full Text]
  30. Reckelhoff J.F., Zhang H., Srivastava K. Gender differences in development of hypertension in spontaneously hypertensive rats: role of the renin–angiotensin system. Hypertension (2000) 35:480–483.[Abstract/Free Full Text]
  31. Leung P.S., Wong T.P., Chung Y.W., Chan H.C. Androgen dependent expression of AT1 receptor and its regulation of anion secretion in rat epididymis. Cell Biol Int (2002) 26:117–122.[CrossRef][Web of Science][Medline]
  32. Ogut O., Brozovich F.V. Regulation of force in vascular smooth muscle. J Mol Cell Cardiol (2003) 35:347–355.[CrossRef][Web of Science][Medline]
  33. Somlyo A.P., Somlyo A.V. Ca2+ sensitivity of smooth muscle and non-muscle myosin II: modulated by G proteins, kinases, and myosin phosphatase. Physiol Rev (2003) 83:1325–1358.[Abstract/Free Full Text]
  34. Faraci F.M., Lamping K.G., Modrick M.L., Ryan M.J., Sigmund C.D., Didion S.P. Cerebral vascular effects of angiotensin II: new insights from genetic models. J Cereb Blood Flow Metab (2006) 26:449–455.[CrossRef][Web of Science][Medline]
  35. Cavarape A., Endlich N., Assaloni R., Bartoli E., Steinhausen M., Parekh N., et al. Rho-kinase inhibition blunts renal vasoconstriction induced by distinct signaling pathways in vivo. J Am Soc Nephrol (2003) 14:37–45.[Abstract/Free Full Text]
  36. Sharpe C.C., Hendry B.M. Signaling: focus on Rho in renal disease. J Am Soc Nephrol (2003) 14:261–264.[Free Full Text]
  37. Uehata M., Ishizaki T., Satoh H., Ono T., Kawahara T., Morishita T., et al. Calcium sensitization of smooth muscle mediated by a Rho-associated protein kinase in hypertension. Nature (1997) 389:990–994.[CrossRef][Medline]
  38. Jin L., Ying Z., Hilgers R.H., Yin J., Zhao X., Imig J.D., et al. Increased RhoA/Rho-kinase signaling mediates spontaneous tone in aorta from angiotensin II-induced hypertensive rats. J Pharmacol Exp Ther (2006) 318:288–295.[Abstract/Free Full Text]
  39. Jackson E.K., Andresen B.T., Seasholtz T.M., Zhu C., Romero G.G. Enhanced activation of RhoA by angiotensin II in SHR preglomerular microvascular smooth muscle cells. J Cardiovasc Pharmacol (2005) 45:283–285.[CrossRef][Web of Science][Medline]
  40. Wingard C.J., Johnson J.A., Holmes A., Prikosh A. Improved erectile function after Rho-kinase inhibition in a rat castrate model of erectile dysfunction. Am J Physiol Regul Integr Comp Physiol (2003) 284:R1572–R1579.[Abstract/Free Full Text]
  41. Reckelhoff J.F., Zhang H., Granger J.P. Decline in renal hemodynamic function in aging SHR: role of androgens. Hypertension (1997) 30:677–681.[Abstract/Free Full Text]
  42. Ruperez M., Sanchez-Lopez E., Blanco-Colio L.M., Esteban V., Rodriguez-Vita J., Plaza J.J., et al. The Rho-kinase pathway regulates angiotensin II-induced renal damage. Kidney Int Suppl (2005) 99:S39–S45.[Medline]
  43. Nishikimi T., Akimoto K., Wang X., Mori Y., Tadokoro K., Ishikawa Y., et al. Fasudil, a Rho-kinase inhibitor, attenuates glomerulosclerosis in Dahl salt-sensitive rats. J Hypertens (2004) 22:1787–1796.[CrossRef][Web of Science][Medline]
  44. Ishikawa Y., Nishikimi T., Akimoto K., Ishimura K., Ono H., Matsuoka H. Long-term administration of Rho-kinase inhibitor ameliorates renal damage in malignant hypertensive rats. Hypertension (2006) 47:1075–1083.[Abstract/Free Full Text]
  45. Sun G.P., Kohno M., Guo P., Nagai Y., Miyata K., Fan Y.Y., et al. Involvements of Rho-kinase and TGF-beta pathways in aldosterone-induced renal injury. J Am Soc Nephrol (2006) 17:2193–2201.[Abstract/Free Full Text]
  46. Hirooka Y., Shimokawa H. Therapeutic potential of Rho-kinase inhibitors in cardiovascular diseases. Am J Cardiovasc Drugs (2005) 5:31–39.[CrossRef][Medline]

Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?



This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow E-letters: Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when E-letters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Song, J.
Right arrow Articles by Martin, D. S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Song, J.
Right arrow Articles by Martin, D. S.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?