INTRODUCTION

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ANDROGENS are recognized as strong modulators of male sexual behavior although the exact role of androgens in the maintenance of erectile responsiveness remains controversial (20, 22). Castration has been found to decrease the erectile responses to a variety of stimuli while androgen replacement reversed these effects (1, 26, 38). In animal models of erectile function the intracavernosal pressure response to ganglionic stimulation is suppressed when administered antiandrogenic therapy or after surgical castration and restored with the androgen replacement therapies (4, 5, 16, 34).

An essential requirement for normal erectile function is the relaxation of the cavernosal sinuses and arterial smooth muscle. This relaxation is a complex interplay of messenger molecules that are capable of shifting the balance of smooth muscle tone from constricted to relaxed. Castration has been associated with alteration in the expression and action of the major relaxation pathway element nitric oxide (NO) and its generating enzyme NO synthase (NOS) (4, 14-16, 28). Such alterations are assumed to be major contributors to the loss of erectile function. Additionally, there are reported changes in the sensitivity of the sympathetic pathway response and adrenergic vasoconstrictor effects increasing smooth muscle tone contributing to the depressed erectile response (27).

While most studies examining the role of androgens on erectile function have focused on the impact of androgens on the NO signaling system, few studies have examined other regulators of erectile function. We were particularly interested in the impact of the loss of androgens on the vasoconstrictor action of the RhoA/Rho-kinase pathway. The constricted state of penile vasculature is considered to be mediated by release of norepinephrine, endothelin-1, and a host of other vasoconstrictors (2). These agents bring about vasoconstriction by elevating intracellular calcium and activating myosin light-chain kinase, resulting in myosin phosphorylation and crossbridge activation. Additionally, a calcium sensitization process is activated through agonist activation of heterotrimeric G protein-coupled receptors, activation of RhoA through exchange of GTP for GDP, and dissociation from a guanine nucleotide dissociation inhibitor (GDI). The activated RhoA then activates Rho-kinase, which inhibits myosin light-chain phosphatase, resulting in a net increase in myosin phosphorylation and force at constant calcium (31, 32).

We have recently demonstrated that the vasomotor activity of the penile circulation was under the influence the RhoA/Rho-kinase pathway, which has a very strong vasoconstrictor effect, and its inhibition results in substantial relaxation and an augmented erectile response (8, 18, 19). Reported here are results of experiments testing the hypothesis that an active Rho/Rho-kinase pathway contributes to the reduced erectile response after castration due to an upregulation of RhoA/Rho-kinase protein levels, and inhibition of the Rho-kinase restores erectile function by relaxing cavernosal smooth muscle.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Animals. Male Holtzman rats (90-120 days of age, 300-325 g, Harlan Laboratories, Indianapolis, IN) were used in all experiments. Animals were divided into three experimental groups: 1) intact: no surgery and served as control; 2) castrate: surgically castrated, implanted with a cholesterol pellet, and allowed to recover for 7-10 days; and 3) testosterone: surgically castrated, with testosterone replacement therapy via a subcutaneous pellet implantation for 7-10 days.

Erectile response measurements. Rats were anesthetized with an intramuscular injection of ketamine (87 mg/kg body wt) plus xylazine (13 mg/kg), and anesthesia was maintained on supplemental ketamine as needed. The left carotid artery was cannulated for continuous monitoring of mean arterial pressure (MAP). The shaft of the penis was freed of skin and fascia, and the right corpus cavernosum was cannulated by insertion of a 30-gauge needle connected to a pressure transducer, permitting continuous monitoring of corpus cavernosal pressure (CCP). The left corpus cavernosum was cannulated with 30-gauge needles attached to 10-µl syringes via short lengths of PE-10 tubing and used for administration (intracavernosal injection) of vasoactive drugs. The abdominal cavity was opened, exposing the right major pelvic ganglion (MPG, contains autonomic nerve fibers that innervate the cavernosal vascular tissue). Platinum bipolar electrodes were positioned on the MPG, and their position was adjusted during stimulation until a maximal voltage-induced response was achieved. During the experiment, stimulatory voltages applied to the MPG ranged from 1 to 5 V delivered in 5-ms pulses at a frequency of 12 Hz. The duration of stimulation was for 1-2 min with rest periods of 2-3 min between subsequent stimulations. All pressure data were collected for analysis using Polyview data-acquisition software (AstroMed, Grass Instrument Division).

Isolated cavernosal tissue force measurements. Cavernosal strips were prepared by amputating the distal portion of the penis and then removal of the corpus spongiosum and dorsal vein. Strips were bathed in a physiological salt solution (PSS) at pH 7.4, 37°C bubbled with breathing air. The PSS was composed of (in mM) 140.0 NaCl, 5.0 KCl, 1.6 CaCl₂, 1.2 MgCl₂, 1.2 Na₂HPO₄, and 5.6 D-glucose. Tissue resting force was set to 500 mg by a series of stretches and length releases, followed by a period of stress relaxation. Setting the tissue to a preset resting force of 500 mg was found to correlate to an optimal length-force generation in response to maximal K⁺ depolarization (data not shown). All tissues were contracted by the addition of 109 mM K⁺ physiological saline solution (KPSS). KPSS was prepared by stoichiometric substitution of KCl for NaCl in PSS. Tissues were depolarized for 10 min and then relaxed with repeated washes of PSS at 10-min intervals before the start of any experimental protocol. The collected results were used to construct cumulative dose-response curves for the -adrenergic agonist phenylephrine (PE) (0.1-100 µM) or the Rho-kinase inhibitor Y-27632 (0.3-30 µM). The relaxation curves with Y-27632 required the strips to be precontracted with 10 µM PE. All dose-response curves were constructed in the presence of inhibitors of the NO pathway (10 µM N-nitro-L-arginine methyl ester).

Immunoblot analysis of cavernosal RhoA and Rho-kinase protein expression. Cavernosal strips (cleaned of the corpus spongiosum and dorsal vein) were frozen in dry ice-acetone slurry and then pulverized in liquid nitrogen. Tissues were homogenized in cold radioimmunodetection buffer that contained 50 mM Tris · HCl (pH 7.4), 1.0% NP-40, 0.25% Na-deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, 1 µg/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml pepstatin, 1 mM Na₃VO₄, and 1 mM NaF. Samples were centrifuged (1,000 g, 4°C, 10 min), and the supernatant was collected for protein quantification and immunoblot analysis. In control experiments, no RhoA or Rho-kinase immunoreactivity was found in the 1,000-g pellet. Equal amounts of protein (100 µg total protein/lane) were loaded and resolved by 10% (Rho-kinase) or 15% (RhoA) SDS-PAGE (at room temperature overnight). Rat brain extract served as a positive control. Proteins were transferred to a nitrocellulose membrane (Immobilon-P, Millipore) using a Bio-Rad Mini-Protean III apparatus (2 h at 4°C) in the presence of 25 mM Trizma base, 191.8 mM glycine, and 20% methanol. The nitrocellulose membrane was then incubated with 5% skimmed milk in phosphate-buffered saline (30 min, room temperature). After blocking, the membrane was incubated overnight (4°C) with primary goat polyclonal antibody to the carboxy terminus of Rho-kinase (1:500 dilution, ROCK-2, Transduction Labs) or primary monoclonal antibody to RhoA, (1:100 dilution, Transduction Labs) and subsequently incubated (2 h at 4°C) with a rabbit anti-mouse antibody (1:500 dilution, Transduction Labs). Antibody-bound protein was visualized using an ¹²⁵I-protein A incubation (2 h at room temperature) (13). Rho-kinase protein expression corresponded to a band in the 170-kDa range while RhoA protein expression corresponded to a band in the 25-kDa range. Total protein determinations were accomplished using a Micro bicinchoninic acid protein assay (Pierce Biotechnology, Rockford, IL)

Drugs. The present study utilized a specific inhibitor of Rho-kinase, Y-27632, generously supplied by the Mitsubishi Pharma Group (Osaka, Japan). Testosterone was purchased from Steraloids (Newport, RI). Cholesterol and PE were purchased from Sigma Chemical (St. Louis, MO).

Data analysis and statistics. Raw force responses were recorded digitally with POLYview data acquisition (Astro-Med, West Warwick, RI). Postacquisition analysis included conversion of force (in g) to stress (mN/mm²). Force was converted to stress values (force/cross-sectional area) by the following equation: {[force (mg) × 0.0987]/area}/1.055 (density conversion), where area is calculated from wet weight (mg)/length (mm). Dose-response profiles were constructed for the individual traces for each experimental condition. Normalized stress was calculated by dividing the measured stress level by the maximal stress level measured during the construction of the dose-response profile. The profiles were fit by Sigma Plot (SPSS Science, Chicago, IL), using a Hill fit protocol, allowing for the report of the estimated EC₅₀s. Data were presented as means ± SE. Statistical differences were determined by ANOVA followed by Bonferroni's complementary analysis, where relevant, and Student's t-test using the SigmaStat Analysis Program (SPSS Science, Chicago, IL). A P value of <0.05 was considered to be significant.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Effect of castration and testosterone-treatment on the voltage-dependent erectile response. To examine the voltage-dependent erectile response, the MPG was stimulated over a range of 1-5 V while MAP and CCP were recorded. The average value of CCP was divided by the average MAP for each stimulus level to provide a quantitative assessment of the erectile response. Ganglionic stimulation resulted in a voltage-dependent increase in CCP/MAP, in accordance with previously published findings (9, 21, 37). Surgical castration of male rats (10 days postsurgery) resulted in a depressed erectile response to voltage stimulation of the major pelvic ganglion compared with intact age-matched animals (Fig. 1). Statistically significant depression in the erectile responses was seen in the castrate group at voltages > 3 V (Fig. 1B, left). Testosterone treatment (surgical castration with implantation of a 50% testosterone-50% cholesterol subcutaneous pellet) restored the voltage-dependent erectile response to that of intact animals (Fig. 1, A and B, left). Castration and testosterone treatment had no effect on MAP or CCP before MPG stimulation (Table 1).

View larger version (25K): [in this window] [in a new window]   Fig. 1.   Voltage-dependent erectile response of intact, castrate (Cast), and testosterone-treated (Testo) rats before and after Rho-kinase inhibition. A: representative traces of the erectile response [corpus cavernosal pressure (CCP)/mean arterial pressure (MAP)] to graded voltages applied to the major pelvic ganglion before and after intracavernosal injection of the Rho-kinase inhibitor Y-27632. B: erectile responses for each applied voltage (based on the average value for 2 min of stimulation at a given voltage). Mean responses ± SE are reported; n = 5-6 animals/experimental group. Left: erectile responses from animals before administration of 5 µl 20 nM Y-27632. Right: erectile responses from the same animals 5 min after administration of Y-27632. * Statistical significance from intact values, P < 0.05.

Effect of Y-27632 on the voltage-dependent erectile response. We have previously established the importance of Rho-kinase in the maintenance of penile vasoconstriction in the normal adult rat. We sought to examine the effects of Rho-kinase inhibition on the erectile response in hypogonadal rats, hypothesizing that a loss in androgen-dependent erectile function was due to upregulation of RhoA/Rho-kinase signaling and could therefore be reversed by inhibition of Rho-kinase activity. Injection of the selective Rho-kinase antagonist Y-27632 (5 µl, 20 nM) into the left corpus cavernosum sinuses resulted in an increase in CCP/MAP in all experimental groups without MPG stimulation (Fig. 1, A and B, right). We found that the intracavernosal injection of Y-27632 had a small but not significant depression in MAP in all experimental groups, while producing a significant threefold elevation in CCP in both castrate and testosterone-treated groups without MPG stimulation (Table 1). The doubling in CCP with Y-27632 injection in intact animals was close to significance with a P value of 0.06 and likely reflects a small sample size. However, each animal demonstrated an elevation in CCP upon injection of Y-27632.

-Adrenergic-mediated contractile response of isolated cavernosal tissue. We examined the concentration-dependent contractile response of isolated corpus cavernosum to the cumulative addition of the -adrenergic agonist PE. Cavernosum from intact and testosterone-treated animals displayed a biphasic response to increasing concentration of PE, rising from 0.1 to 10 µM, and then declining at concentrations >10 µM. Cavernosal strips from castrate animals displayed a monotonic response with a plateau in stress >10 µM and a maximal stress response that was significantly larger than the intact and testosterone-treated responses (Fig. 2A). The increased stress response with castration was also associated with a significant rightward shift in the PE dose-response profile as reflected by changes in the average EC₅₀ values (intact 0.299 ± 0.057 µM; testosterone treated 0.209 ± 0.075 µM; castrate, 0.729 ± 0.092 µM; P < 0.05, castrate vs. intact) (Fig. 2B).

View larger version (18K): [in this window] [in a new window]   Fig. 2.   Stress-generation responses of isolated cavernosal strips to increasing concentration of the -adrenergic agonist phenylephrine (PE). A: increases in PE concentration were associated with increased stress generation in all experimental groups over the 0.1- to 3.0-µM range. Castration was associated with a significant rightward shift of concentration-response relationship, and testosterone treatment returned the concentration-response relationship to that of the intact (B). * Statistical significance from intact values, P < 0.05; n = 4-6. Labeled EC50 values are based on mean data.

Effect of Y-27632 on -adrenergic-mediated contraction of isolated cavernosal tissue. To examine the effect of Rho-kinase inhibition on the maintenance of adrenergic-mediated stress, isolated corporal strips were first contracted with 10 µM PE and subsequently relaxed with increasing concentrations of Y-27632. The increasing concentrations of Y-27632 resulted in a graded relaxation of corporal strips from all experimental groups. There was a significant higher stress level in response to 10 µM PE prestimulation in corpora from castrate animals (Fig. 3A). However, the sensitivity to Rho-kinase inhibition was not different between the experimental groups with average EC₅₀ values of 0.909 ± 0.467 (intact), 1.259 ± 0.369 (castrate), and 0.964 ± 0.397 µM (testosterone treated) (Fig. 3B).

View larger version (15K): [in this window] [in a new window]   Fig. 3.   Relaxation of isolated cavernosal strips to graded inhibition of Rho-kinase by Y-27632. A: increases in Y-27632 concentration were associated with reductions in stress induced by 10 µM PE in all experimental groups over the 0.3- to 30-µM range. Castration and testosterone treatment were not associated with any significant shift in the concentration-response relationship (B). * Statistical significance from intact values, P < 0.05; n = 4-6. Labeled EC50 values are based on mean data.

Effect of castration and testosterone treatment on expression of RhoA and Rho-kinase levels in isolated cavernosal tissues. We have demonstrated that castration results in a depressed erectile response and increased constriction of corporal cavernosal tissues to the -adrenergic agonist PE. We sought to examine the impact of castration and testosterone treatment on the expression pattern of the enzyme Rho-kinase and its upstream regulator RhoA in the corporal tissues to test the hypothesis that increases in the of RhoA and/or Rho-kinase protein levels contribute to increased constrictor activity and decreased erectile response. Castration was associated with a significant increase in the amounts of both RhoA and Rho-kinase in the cavernosal tissues (Figs. 4A and 5A). Testosterone treatment of castrated rats lowered the levels of RhoA/ Rho-kinase protein detected but did not return them to intact levels. Castration resulted in a >15-fold increase in the amount of RhoA detected by Western blotting (Fig. 4B). The mean optical density units for RhoA, normalized for the total protein levels, were 6.3 ± 1.0 (intact), 74.3 ± 8.1 (castrate; P < 0.05 vs. intact), and 57.8 ± 10.5 (testosterone treated; P < 0.05 vs. intact; n = 4-9). A similar pattern of protein detection was seen with Rho-kinase. Castration resulted in a greater than twofold increase in the amount of Rho-kinase detected by Western blotting (Fig. 5B). The mean optical density units for Rho-kinase normalized for the total protein levels were 16.0 ± 3.6 (intact), 27.5 ± 6.6 (castrate; P < 0.05 vs. intact), and 24.9 ± 12.5 (testosterone treated; P < 0.05 vs. intact; n = 3-6). The 70-kDa band seen in the RhoA and Rho-kinase blots was determined to be nonspecific staining.

View larger version (35K): [in this window] [in a new window]   Fig. 4.   Immunodetection of RhoA in cavernosal tissue from intact, castrate, and testosterone-treated animals. A: representative Western blots for RhoA from tissue homogenates of isolated cavernosal strips. In each lane 100 µg of total protein was loaded. B: graph presents quantitation of fold change in optical density of 23-kDa band, which corresponded to RhoA. * Statistical significance from intact levels at P < 0.05; n = 4-9 animals.

View larger version (32K): [in this window] [in a new window]   Fig. 5.   Immunodetection of Rho-kinase in cavernosal tissue from intact, castrate, and testosterone-treated animals. A: representative Western blots for Rho-kinase from tissue homogenates of isolated cavernosal strips. In each lane 100 µg of total protein was loaded. B: graph presents quantitation of fold change in optical density of 180-kDa band, which corresponds to Rho-kinase. * Statistical significance from intact levels at P < 0.05; n = 3-6 animals.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The findings reported from these studies demonstrate that castration decreased erectile response to ganglionic stimulation and increased constrictor responsiveness to PE stimulation, which was reversed by inhibition of the calcium-sensitizing pathway involving Rho-kinase. Furthermore, the decreased erectile response and increased constrictor response was associated with increased RhoA and Rho-kinase protein levels, which could be lowered by testosterone replacement.

Although the exact role of androgens in the maintenance of erectile responsiveness remains controversial, it is known that erectile function (3) and nocturnal penile tumescence episodes are suppressed in severely hypogonadal men (10, 11, 33). While most of the studies on the role of androgens on erectile function have focused on the impact of androgens on NO or its signaling pathway, little emphasis has been placed on other regulators of erectile function. We were particularly interested in the impact of the loss of androgens on the vasoconstrictor action of the RhoA/Rho-kinase pathway.

We hypothesized that an increase in cavernosal Rho-kinase and its upstream regulator RhoA contributes to the erectile dysfunction seen in castrate rat. Intracavernosal injection of the Rho-kinase inhibitor Y-27632 (12, 35) (20 nM) resulted in an increase in CCP/MAP, which restored the erectile response of castrate animals to levels equivalent to intact and testosterone-treated castrates (Fig. 1). The suppressed erectile response was associated with an increased expression of both Rho-kinase and its upstream regulator RhoA (Figs. 4 and 5).

Previous studies using the rat model have found a significant suppression of ganglion-induced erectile responses (CCP/MAP) with castration, suggesting a depressed NO release (26-28). We found in these studies that voltage stimulation of the major pelvic ganglion induced an increase in CCP/MAP, which was significantly suppressed in the 10-day castrate male rat, compared with intact age-matched controls (Fig. 1). The in vitro constrictor response to the -adrenergic agonist PE was elevated in corpus cavernosal strips from the castrate animals (Fig. 2). This response differs from that reported by Alcorn and coworkers (1), who examined the contractile response to 100 µM norepinephrine and found no difference between intact, castrate, and testosterone-treated animals. This discrepancy may reflect a difference in the tissue responsiveness to the agonist of choice, its concentration, or to the duration of castration protocol employed. Our studies show a significant reduction in contractile force to 100 µM levels of PE, which may reflect the receptor desensitization to this high agonist concentration. Using a rat castrate model similar to the one employed in these studies, Reilly and coworkers (27) found that a portion of the depressed erectile response associated with castration was attributed to an increased -adrenergic responsiveness. These later results are confirmed by our observation of an increased constrictor response in vitro in cavernosal tissues from castrated animals.

In addition to the increased maximal stress generation, we found the response profile to the ₁-adrenergic agonist PE was shifted to the right in castrated animals (Fig. 2B) and was restored to the intact profile with testosterone treatment. Such a response could reflect a change in receptor population or subtype. A recent review by Andersson (2) cites studies that demonstrated changes in receptor populations with erectile disorders and that some of the most recently identified ₁-receptor subtypes have different affinities for selective antagonists. Traish and coworkers (34) reported that castration reduced the expression of ₁-adrenergic receptor in cavernosal tissue, and whose reduction was prevented or reversed by testosterone replacement. Our data are consistent with a possible change in receptor population or subtype as we observed a change in maximal response and a shift in the calculated EC₅₀ values (effective concentration to invoke 50% of the maximal response). In addition, our results suggest that with the loss of androgen through castration, there is an increased calcium sensitization effect, resulting in the augmented force generation seen in isolated strips.

It is tempting to speculate that the increased responsiveness to -adrenergic stimulation in this model may be associated with an increased activity of Rho-kinase through lowered NO production. There is evidence for an inhibitory effect of NO on the RhoA/Rho-kinase signaling pathway such that NO activation of protein kinase G (PKG) results in phosphorylation of RhoA and its inhibition (17, 29). In the castrate model where there is depressed NO production, subsequent PKG activity may be depressed and unable to curtail Rho-kinase activity. Some evidence is reported to suggest that exogenously applied NO can suppress the RhoA/Rho-kinase pathway action and aid in normal erection (17, 19). If such a mechanism of action of NO occurs, then in conditions of low NO, the resulting maintained Rho-kinase activity levels would indirectly inhibit myosin phosphatase and maintain cavernosal smooth muscle tone as seen in this model of erectile dysfunction. Alternatively, the balance between activities or expression levels of elements of calcium-dependent and calcium-sensitizing pathways regulating contracture may be altered by castration.

Interestingly, testosterone treatment of castrated animals restores the erectile responsiveness. Several groups report that testosterone acts to maintain the nNOS expression or NO levels (1, 6, 15, 16, 28, 30, 38). This may result in not only restoration of an NO-mediated relaxation via increased Ca²⁺ sequestration and decreased myosin light-chain kinase activity but also through the inhibition of Rho-kinase and maintained myosin phosphatase activity.

The calculated EC₅₀ values of isolated corpora cavernosal strips to Rho-kinase inhibition in all experimental groups from our studies were similar with reported values near 1 µM. Our results are in line with those reported in the literature, including those of Wang et al. (36) for rabbit permeabilized corpora cavernosal tissues, whereas our values were slightly lower or equal to those reported by Rees et al. (24) in intact tissues of human (2.2 µM) and rabbit (1.0 µM) cavernosum. In light of the similar EC₅₀ values but elevated levels of RhoA and Rho-kinase detected in castrate tissues, our results may suggest a difference in the total amount of active RhoA or Rho-kinase between experimental groups is responsible for the observed force and erectile behavior. However, a direct measure of RhoA activation or Rho-kinase activity needs to be made to confirm this conclusion.

We have previously demonstrated the importance of the calcium-sensitizing enzyme Rho-kinase in the maintenance of penile detumescence and found that selective inhibition of Rho-kinase results in sustained increase in the CCP/MAP ratio. Reports from others have demonstrated the impact of Rho-kinase activity on the erectile response in models of hypertension (7). Several groups have identified the expression of Rho-kinase in cavernosal tissue from other species (25, 36).

Our studies have shown the Rho-kinase inhibitor Y-27632 initiates vasodilatation by directly inhibiting Rho-kinase and not by stimulating the release of NO (8). For this reason, Rho-kinase inhibition may be an option for use in the treatment of erectile dysfunction in patients lacking sufficient androgen levels to maintain normal NO production. Furthermore, other approaches to inhibition of the RhoA/Rho-kinase signaling could include altering the activity of associated molecules like GDI that are essential for the activity of the pathway.

Altogether, our findings support the hypothesis that erectile dysfunction associated with hypogonadism is in part mediated through an increased RhoA/Rho-kinase pathway, preventing relaxation of penile smooth muscle tone and erection. This has particular significance as most previous studies have examined altered NO relaxation mechanisms and not mechanisms associated with maintenance of smooth muscle tone.