¹Department of Urology, Chonbuk National University Medical School, Chonju, South Korea

²Department of Physiology, Chonbuk National University Medical School and Chonbuk National University Institute for Medical Sciences and Research Institute of Clinical Medicine of Chonbuk National University Hospital, Chonju, South Korea

³Pohang University of Science and Technology, Pohang, South Korea

Correspondence to: J K Park, Department of Urology, Chonbuk National University Medical School, 2-20 Keum-Am-Dong-San, Chonju 561-712, South Korea. E-mail: rain@moak.chonbuk.ac.kr

Isometric tension measurement using a selective Rho-kinase inhibitor (+)- (R)-trans4-(1-aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide (Y-27632) and a selective myosin light chain kinase (MLCK) inhibitor 1-(5-iodonaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine hydrochloride (ML7) were used in rabbit clitoral cavernosum smooth muscle (CSM). N^G-nitro-L-arginine methyl ester (L-NAME) was used to evaluate the relationship between NO release and Rho-kinase. Y-27632 significantly attenuated contractions induced by ANG II, dose-dependently. However, ML7 did not affect the contractile response to ANG II except in the high concentrations of ML7. Y-27632 inhibited contraction with phenylephrine (PhE), but ML7 did not inhibit contraction with PhE. Nitric oxide synthase inhibitor (NAME) did not affect the Y-27632-induced relaxation in the pre-contracted strip with PhE. The present study demonstrates that G-protein-coupled increase in myofilament Ca²⁺ sensitivity mediated through the RhoA/Rho-kinase signal pathway is involved in the control by ANG II of the clitoral CSM tone. RhoA/Rho-kinase pathway acts in the ANG II-induced contraction independently of the NO pathway.

International Journal of Impotence Research (2002) 14, 472-477. doi:10.1038/sj.ijir.3900911

clitoral cavernosum smooth muscle; angiotensin II; RhoA/Rho-kinase; myosin light chain; Ca²⁺ sensitization

Introduction

The tone of clitoral cavernosal artery and smooth muscle is regulated by the balance between the release of the vasoconstrictors and smooth muscle relaxants. The physiological actions of angiotensin (ANG) II are many. Peripherally, the actions of ANG II are intimately involved in hemodynamic and extracellullar fluid regulation.¹ ANG II receptors have been characterized in the mammalian cavernosum tissues. ANG II receptors are divided into type 1 (AT1) and type 2 (AT2).² ANG II is an important vasoconstrictor and increases penile and clitoral cavernosal smooth muscle (CSM) tone via the AT1 receptor.^2,3 ANG II receptor blocker has been shown to be highly effective in increasing the penile cavernosal pressure, resulting in penile erection.⁴

Angiotensin-converting enzyme (ACE) inhibitor and AT1 receptor blocker are potentially useful for the treatment of hypertension. Those medications did not increase the frequency of sexual problems, although many commonly used antihypertensive agents interfere with sexual function in men and women.⁵ The results showed that there was a relationship between renin angiotensin system (RAS) and erectile function in men and women.

It is well known that smooth muscle contraction is primarily regulated by the level of 20 kDa myosin light chain (MLC) phosphorylation. MLC is dephosphorylated by the Ca²⁺-independent myosin light chain phosphatase (MLCP). The extent of MLC phosphorylation is determined by the balance of activities of the enzymes myosin light chain kinase (MLCK) and MLCP. Activation of MLCK is dependent on intracellular concentration of free cytosolic Ca²⁺ whereas inhibition of MLCP increases the level of phosphorylated MLC and helps to develop and/or maintain tension in smooth muscle at a constant Ca²⁺ concentration through the G-protein coupled pathway, which is defined as Ca²⁺ sensitization (Figure 1).^6,7,8 Conversely, relaxation of vascular smooth muscle results from decrease in cytosolic Ca²⁺ concentration and/or reduced calcium sensitivity of the contractile apparatus. Increases in activity of MLCP by blockade of RhoA/Rho-kinase-mediated signaling pathway attenuated the contractile responses via the inhibition of Ca²⁺ sensitizing mechanism.⁹ The small G-protein RhoA is involved in agonist-induced Ca²⁺ sensitization. RhoA activates Rho-kinase, resulting in phosphorylation of the myosin-binding subunit of MLCP and reduces MLCP activity.¹⁰ RhoA proteins are primarily found in the cytosol fraction of unstimulated cells or tissues, as the GDP-bound (inactive) form. When cells or tissues are stimulated, the small G-protein translocates from the cytosol to the membrane fraction as the GTP-bound (active) form.^11,12,13

(+) - (R) - trans - 4 - (1 - aminoethyl) - N - (4 - pyridyl)cy-clohexanecarboxamide (Y-27632), a selective Rho-kinase inhibitor, enhanced relaxation occurring in the penile and bronchial smooth muscle.^8,9 It was reported that the increase of blood pressure and the decrease of cavernosum pressure were potently inhibited by Y-27632.^8,14 However, little is known about the role of Rho-kinase in ANG II-induced smooth muscle contraction in the clitoris. We evaluated the effect of Y-27632 in relation to ANG II in the clitoral CSM.

Materials and methods

Preparation of isolated clitoral CSM strips

Clitoral erectile tissue was obtained from female New Zealand white rabbits (2-2.5 kg), as described previously.³ Briefly, female rabbits were anesthetized with sodium thiopental (30 mg/kg intravenously) and exsanguinated. The entire clitoris including vagina was excised rapidly. The vaginal wall was dissected free from the clitoral body. The clitoral cavernosum tissue was then carefully dissected free from the surrounding tunica albuginea under dissecting microscope and two strips along the longitudinal axis (about 0.5´0.5´5 mm) were obtained. During the preparation, care was taken not to damage the functional endothelium or overstretch the tissue.

Measurement of isometric tension and recording of mechanical activity

A strip of the rabbit clitoris CSM (n=58) was vertically placed in 2 ml organ chamber with one end connected with a cotton thread to the prong of force transducer (Grass Research Polygraph, Model 79H, W. Warwick. RI, USA), and the other end secured with a cotton thread to a holder for isometric tension measurements.^2,3 The bath chambers contained the appropriate Hepes buffer solution (37°C) and were constantly aerated with 100% O₂. Isometric force was measured and recorded using a polygraph (Grass Research Polygraph, Model 79H, W. Warwick, RI, USA). After mounting, strips were equilibrated for 60 min with several adjustments of length until a baseline force stabilized at 1 g and oxygenated medium was replaced every 20 min.

Contractile responses to ANG II

In the first series of experiments following equilibration, the preparations were initially stimulated with 10^-5 M L-phenylephrine solution (PhE, Sigma Chemical Company, St Louis, MO, USA) to obtain a reference response. When contractile responses to the PhE had reached a plateau, we added acetylcholine (ACh, Sigma Chemical Company, 10^-7-10^-5 M) to confirm the presence of endothelium. After confirming the relaxations, strips were rinsed with Hepes buffer. ACh induced marked reduction of the PhE-induced tone, taken as evidence that a significant amount of functional endothelium was present. After the test for endothelium, strips were rinsed three times with Hepes buffer solution and allowed to return to baseline tension and re- equilibrate for 10 min before subsequent exposure to ANG II.

In the second series of experiments, we examined the effect of ANG II (Sigma Chemical Company, 10^-9-10^-7 M). Effects of agents were summed and recorded after contractions reached the highest amplitude.

Effects of Y-27632 and ML7 on the responses to ANG II

After response to the ANG II was finished, experiments were performed to investigate effect of pre-incubation with Y- 27632 (Tocris Cookson Ltd, UK, 10^-8-10^-5 M, n=8) and 1-(5-iodonaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine hydrochloride (ML7, BIOMOL, Plymouth Meeting, PA, USA, 10^-8-10^-5 M, n=8) on cumulative dose-dependent contractile response (DDCR) to ANG II in the same strips. Y-27632 and ML7 were added at least 30 min belore ANG II was given.

Effects of the pre-incubation with Y-27632 and ML7 in the contractions induced by PhE

We examined the effect of the pre-incubation with Y-27632 or ML7 (n=7) on the contractions induced by PhE. In separate experiments (n=7), after pre-incubation with Y-27632 (10^-8-10^-5 M) for 30 min, a single dose of PhE (10^-5 M) was added to the organ bath. In separate experiments (n=7), the effects of ML7 (10^-9-10^-4 M) were evaluated in a similar protocol.

Effects of the Y-27632 and ML7 in the pre-contracted strips with PhE

We examined the effect of cumulative Y-27632 and ML7 in the pre-contracted strips with PhE. In separate experiments (n=7), after confirming the plateau of the PhE-induced contraction, we added Y-27632 (10^-8-10^-5 M) to the tissue baths. In separate experiments (n=7), the effects of ML7 (10^-9-10^-4 M) were evaluated in a similar protocol.

Relation of nitric oxide (NO) signal transduction with RhoA/Rho-kinase pathway

We investigated cross-talking between NO signal transduction and RhoA/Rho-kinase MLCP and ML7-MLCK pathway. In separate experiments (n=7), we added Y-27632 to the tissue baths after confirming a recovered plateau of the PhE-induced contraction. Then the strip was washed three times for 10 min. The strip was incubated with N^G-nitro-L-arginine-methyl ester (L-NAME, Sigma Chemical Company, 10^-3 M) for 30 min, after the response to PhE was recovered. After confirming the maintained plateau PhE-induced contraction, we added Y-27632 (10^-8-10^-5 M) to the tissue baths. Effects of agents were summed and recorded after contractions reached the highest amplitude.

Relation of nitric oxide (NO) signal transduction with ML7-MLCK pathway

We investigated cross-talking between NO signal transduction and the ML7-MLCK pathway. In separate experiments (n=7), we added ML7 to the tissue baths after confirming a recovered plateau of the PhE-induced contraction. Then the strip was washed three times for 10 min. The strip was incubated with L-NAME for 30 min, after the response to PhE was recovered. After confirming the maintained plateau PhE-induced contraction, we added ML7 (10^-8-10^-4 M) to the tissue baths. The effects of the agents were summed and recorded after contractions reached the highest amplitude.

Data and statistical analyses

Data are expressed as the mean±s.e.m. Contractile responses to ANG II are expressed as percentages of PhE-induced contractile response. The statistical significance of differences was calculated by one-way analysis of variance (ANOVA) followed by Bonferroni's multiple comparisons test and dose-dependent responses before and after treatment with antagonists were compared by Student's paired-t test. A probability value less than 0.05 was considered significant.

Results

Effect of Ach

PhE (10^-5 M) induced sustained contractions of 0.37±0.06 g. ACh (10^-7-10^-5 M) produced dose- dependent relaxation in the strips pre-contacted with PhE by 19±5.12, 42±6.56 and 61±9.42%, respectively.

Contractile responses to ANG II

ANG II produced DDCR in the clitoral CSM (Figure 2).

Effects of Y-27632 or ML7 on ANG II-induced contraction

We evaluated the influence of pre-treatment of clitoral CSM with Y-27632 (10^-8-10^-5 M) and ML7 (10^-9-10^-4 M) on the contractile responses to ANG II. Y-27632 significantly attenuated contractions induced by ANG II, dose-dependently (Figure 2). However, ML7 did not affect the contractile responses to ANG II except at high concentrations of ML7 (10^-4 M; Figure 3).

Effects of pre-incubation with Y-27632 or ML7 on the response to PhE

Y-27632 (10^-8-10^-5 M) caused concentration-dependent decreases in the contractions induced by PhE of 10^-5 M (Figure 4); however, ML7 did not affect the contractions induced by PhE of 10^-5 M, except at high concentrations of ML7 (10^-4 M; Figure 4).

Effects of Y-27632 or ML7 on the pre-contracted strips with PhE

Y-27632 caused concentration-dependent decreases in the pre-contracted strip with PhE of 10^-5 M (Figure 5A). However, ML7 did not affect the pre-contracted strips with PhE, except at high concentrations of ML7 (10^-4 M; Figure 5B). PhE-induced contractions were mildly inhibited by ML7 concentration-dependently at low doses without statistical significance.

Relationship of NO signal transduction to Rho/Rho-kinase pathway

The pre- incubation with L-NAME (10^-3 M) did not change the cumulative effects of Y-27632 in the strip pre-contracted with PhE (Figure 5A).

Relation of NO signal transduction with ML7-MLCK pathway

The pre-incubation with L-NAME (10^-3 M) did not change the cumulative effects of ML7 in the strip pre-contracted with PhE (Figure 5B), except at high concentrations of ML7 (10^-4 M).

Discussion

The present study indicates that the Rho-kinase signaling pathway is involved in the regulation of ANG II-induced contraction in the clitoral CSM. Abnormal contraction of the clitoral CSM and artery results in decreased blood flow into the clitoris and erectile dysfunction. However, relaxation of the clitoral CSM and artery results in increased blood flow in the clitoris, resulting in erection. The balance between the release of the vasoconstrictors and smooth muscle relaxants is an important factor to maintain the tone of the clitoral CSM.^2,15

Contractile response of the smooth muscle is regulated by the level of MLC phosphorylation. Ca²⁺ sensitization refers to the ability of agonists to enhance smooth muscle contractile force at a given level of Ca²⁺ concentration. Rho-kinase induces contraction through an increase in the phosphorylation of the MLC, independent of the Ca²⁺-calmodulin-dependent MLCK.⁶ Rho-kinase phosphorylates the regulatory subunit of MLCP, inhibiting the activity of MLCP, resulting in smooth muscle contraction.¹⁰ The Ca²⁺ sensitization mediated by Rho-kinase results in an increase in blood pressure and hypertension.¹⁴

Recently, it was shown that ANG II produced by the local renin angiotensin system is involved in the regulation of the contractile response via the AT1 receptor in the penis and clitoris.²³ Binding of the ANG II to the AT1 receptor results in a G-protein-mediated activation of phospholipase C, and probably also phospholipases D and A2, which, in turn, results in phosphoinositide hydrolysis, calcium mobilization, calcium influx and inhibition of adenylate cyclase.^17,18 ACE inhibitor and AT1 receptor blocker are potentially useful for the treatment of hypertension. Those medications did not increase the frequency of sexual problems, although many commonly used antihypertensive agents interfere with sexual function in men and women.⁵ Those results showed that there is a relationship between RAS and erectile function in men and women.

Activation of the AT1 receptor is also known to activate Rho protein.¹⁹ The roles of RhoA and Rho- kinase on ANG II-induced contraction in the clitoral CMS have not been determined. The specific Rho-kinase inhibitor, Y-27632, selectively inhibits smooth muscle contraction by inhibiting Ca²⁺ sensitization.²⁰ The present study demonstrates that pre-treatment with Y-27632 (from 10^-8 M) dose-dependently decreases ANG II-induced contractions. Y-27632 of 10^-5 M abolished ANG II-induced contractions (P<0.00001). Pre-treatment with Y-27632 would increase MLCP activity and decrease MLC phosphorylation. Under such conditions, contractile responses to AT1 receptor agonist ANG II would be attenuated, because contractions occur as a result of increasing phosphorylation of MLC by increased activity of MLCK.¹⁷ Interestingly, Y-27632 counteracted both agonists (PhE and ANG II), suggesting that activation of the RhoA/Rho-kinase signaling pathway is an important mechanism underlying contractile responses to these two agonists in clitoral smooth muscle.

ML7 is a specific inhibitor of MLCK. In this study, ML7 did not inhibit ANG II-induced contraction except in the highest concentration (10^-4 M). This result reinforces the idea that ANG II-induced contraction is induced by a marked increase in Ca²⁺ sensitivity mediated through activation of the RhoA/Rho-kinase signal pathway rather than MLCK phosphorylation in the clitoral CSM. At low doses of ML7, PhE-induced contractions were inhibited by ML7 concentration-dependently. This suggests that MLCK may play a role in PhE-induced contraction, although there was no statistical significance. Although NO is the most important transmitter in initiating and maintaining the erection, the inhibition of vasoconstrictors also has a synergistic effect with the NO in the smooth muscle relaxation.²¹ In rat aorta and penis, NO inactivated RhoA-induced contraction,^22,23 however, Y-27632 caused concentration-dependent relaxation in the rabbit clitoral strip pre-contracted with PhE, which was pre-incubated with L-NAME. This result supposes that Y-27632 causes relaxation independently with NO-induced pathway in the rabbit clitoral CSM.

In conclusion, the present study demonstrates that a selective Rho-kinase inhibitor Y-27632 exhibited a counteracting action against PhE and ANG II in the rabbit clitoral CSM. Therefore, it is suggested that the RhoA/Rho-kinase signal pathway is involved in the regulation of the clitoral CSM tone induced by ANG II.

Acknowledgements

This study was supported by a grant from the Institute of Clinical Medicine of Chonbuk National University Hospital.

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Figure 1 Schematic diagram of clitoral cavernosum smooth muscle cell contraction by RhoA/Rho-kinase. PS, phosphorylation; DPS, dephosphorylation; MLCP, myosin light chain phosphatase; MLCP-℗, phosphorylated MLCP; MLC, myosin light chain; MLC-℗, phosphorylated MLC; MLCK, indicates myosin light chain kinase.

Figure 2 Effect of pre-incubation with (+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide (Y-27632, 10^-8-10^-5 M) on clitoral cavernosum smooth muscle (CSM) contracted by angiotensin (ANG) II (10^-9-10^-7 M, n=8). Each point represents the mean±s.e.m. of percentages of maximal relaxation of the preceding submaximal contractile responses generated by phenylephrine (PhE, 10^-5 M). Y of 10^-8 M significantly attenuated ANG II-induced contraction (P<0.05). Y in high concentrations significantly inhibited ANG II-induced contractions. * P<0.05; ** P<0.01. Y, Y-27632.

Figure 3 Effect of pre-incubation with a selective myosin light chain kinase inhibitor 1-(5-iodonaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine hydrochloride (ML7, 10^-8-10^-4 M) on clitoral CSM contracted with ANG II (10^-9-10^-7 M, n=8). Each point represents the mean±s.e.m. of percentages of maximal relaxation of the preceding submaximal contractile responses generated by PhE (10^-5 M). ML7 did not affect the responses to ANG II, except that high concentrations of ML7 (10^-4 M) significantly inhibited ANG II-induced contractions. * P<0.05; *** P<0.001.

Figure 4 Dose-relative effects of pre-incubation with Y-27632 (10^-8-10^-5 M, n=7) and ML7 (10^-8-10^-4 M, n=7) on clitoral CSM contraction induced by a single addition of PhE (10^-5 M). Each point represents the mean±s.e.m. of percentages of maximal relaxation of the preceding submaximal contractile responses generated by PhE. Pre-incubation of Y-27632 significantly inhibited contraction induced by PhE. Pre-incubation with ML7 did not affect the contraction induced by PhE except at high concentrations of ML7 (10^-4 M).

Figure 5 Effect of Y-27632 (10^-8-10^-5 M, n=7) and ML7 (10^-8-10^-4 M, n=7) on the pre-contracted clitoral CSM with PhE (10^-5 M) and PhE pre-contracted strip, which was pre- incubated with N^G-nitro-L-arginine-methyl ester (L-NAME, 10^-3 M). (A) Cumulative effects of Y-27632 on the pre-contracted strip with PhE and PhE pre-contracted strip, which was pre-incubated with L-NAME for 30 min. (B) Cumulative effects of ML7 on the pre-contracted strip with PhE and PhE pre-contracted strip, which was pre-incubated with L-NAME for 30 min. Y-27632 significantly inhibited the pre-contracted strip with PhE. Relaxations induced by Y-27632 on the pre- contracted strip with PhE were not affected by pre-incubation with L-NAME. ML7 did not significantly attenuate the pre-contracted strip with PhE, however PhE-induced contractions were inhibited concentration-dependentlt at low doses without statistical significance. Each point represents the mean±s.e.m. of percentages of maximal relaxation of the preceding submaximal contractile responses generated by PhE.