Improved erectile function following Rho-kinase inhibition in a rat castrate model of erectile dysfunction

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1 Articles in PresS. Am J Physiol Regul Integr Comp Physiol (February 6, 2003) /ajpregu Final Excepted Version R R1 Improved erectile function following Rho-kinase inhibition in a rat castrate model of erectile dysfunction Christopher J. Wingard 1, John A. Johnson 2, Andre Holmes 1, and Anita Prikosh 1 1 Department of Physiology 2 Department of Pharmacology and Toxicology Medical College of Georgia Augusta GA, USA Correspondence: Dr. Christopher J. Wingard Department of Physiology Medical College of Georgia th Street Augusta, GA Phone: (706) Fax: (706) Cwingard@mail.mcg.edu Running heading: Restoration of erection by Rho-kinase inhibition 1 Copyright (c) 2003 by the American Physiological Society.

2 Abstract Final Excepted Version R R1 Androgens are reported to act as strong modulators of erectile function influencing both nitric oxide and vasoconstrictor signaling. Castration results in a depressed erectile response that is associated with a loss of nitric oxide production and increased responsiveness to constrictive agents. The increased vasoconstrictor response may be a result of an active RhoA/Rho-kinase signaling pathway. We report here results of studies designed to test the hypothesis that inhibition of the Rho-kinase pathway restores erectile function in a castrate model by relaxing the smooth muscle. Mean arterial (MAP) and corpus cavernosal (CCP) pressures were monitored during intracavernosal injection of the Rho-kinase inhibitor Y Castration reduced the maximal erectile response (CCP/MAP) by 33 % and testosteronereplacement restored the response (Intact, ± 0.040; Castrate, ± 0.022; testosterone, ± 0.073). Injection of Y increased CCP in all experimental groups it also left shifted the voltage response curve and increased the maximal CCP/MAP response (Intact, ± 0.091; Castrate, ± 0.081; Testosterone-treated, ± 0.033). Y dose dependently relaxed phenylephrine stimulated cavernosal tissues. Cavernosal tissues showed increased RhoA and Rho-kinase protein levels following castration. Our data supports the hypothesis that an active Rho/Rho-kinase pathway contributes to the reduced erectile response following castration due to an up regulation of RhoA/ Rhokinase protein levels and that inhibition of this pathway may serve as an effective treatment for erectile dysfunction. Keywords: smooth muscle contraction, corpus cavernosum, phenylephrine, testosterone, RhoA 2

3 Introduction Final Excepted Version R R1 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 anti-androgenic therapy or following 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 nitric oxide 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 particular 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 3

4 are results of experiments testing the hypothesis that an active Rho/Rho-kinase pathway contributes to the reduced erectile response following castration due to an up regulation of RhoA/ Rho-kinase protein levels and inhibition of the Rho-kinase restores erectile function by relaxing cavernosal smooth muscle. 4

5 Materials and Methods Final Excepted Version R R1 Animals Male Holtzman rats ( days of age, g, Harlan Laboratories, Indianapolis, IN) were used in all experiments. Animals were divided into three experimental groups: INTACT: no surgery and served as control. CASTRATE: surgically castrated, implanted with a cholesterol pellet, and allowed to recover for 7-10 days. TESTOSTERONE: surgically castrated, testosterone replacement therapy via a subcutaneous pellet implantation for 7-10 days. Testosterone pellets were made in the laboratory using a pellet press. Each animal in the Castrate and Testosterone groups was surgically castrated under anesthesia and implanted with a 3 mm pellet (approximately 5 mg) composed of 100% cholesterol or 50% cholesterol /50 % testosterone. This concentration and duration of treatment has previously been determined to severely reduce or maintain normal serum testosterone levels (23,26). Animals were maintained in an American Association for Accreditation of Laboratory Animal Care facility with animal use protocols and justification approved by the Institutional Committee on Animal Use in Research and Education in accordance with NIH guidelines. Erectile Response Measurements Rats were anesthetized with an intramuscular injection of ketamine (87 mg/kg body weight) plus xylazine (13 mg/kg) and anesthesia was maintained on supplemental ketamine as needed. The left carotid artery was cannulated for continuous monitoring of 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 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 which innervate the cavernosal vascular tissue). Platinum bipolar electrodes were positioned on the 5

6 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 volts delivered in 5 millisecond pulses at a frequency of 12 Hz. The duration of stimulation was for 1-2 minutes with rest periods of 2-3 minutes between subsequent stimulations. All pressure data were collected for analysis using Polyview data acquisition software (AstroMed Inc., 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 at ph 7.4, 37 C bubbled with breathing air. The physiological salt solution (PSS) was composed of the following (in mm): NaCl, 5.0 KCl, 1.6 CaCl 2, 1.2 MgCl 2, 1.2 Na 2 HPO 4, 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 minutes and then relaxed with repeated washes of PSS at 10-minute intervals before the start of any experimental protocol. The collected results were used to construct cumulative dose response curves for the α- adrenergic agonist, phenylephrine (PE) ( µm) or the Rho-kinase inhibitor, Y ( µm). The relaxation curves with Y required the strips to be pre-contracted with 10 µm PE. All dose response curves were constructed in the presence of inhibitors of the nitric oxide pathway (10 µm L- NAME). 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 which contained: 50 mm Tris-HCl (ph 7.4), 1.0% NP-40, 0.25% Na-deoxycholate, 150 mm 6

7 NaCl, 1 mm EDTA, 1 mm PMSF, 1 µg/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml pepstatin, 1 mm Na 3 VO 4, and 1 mm NaF. Samples were centrifuged (1,000 g, 4 o C, 10 minutes), and the supernatant collected for protein quantification and immunoblot analysis. In control experiments no RhoA or Rhokinase immuno-reactive was found in the 1,000 g pellet. Equal amounts of protein (100 µg total protein per lane,) were loaded and resolved by 10% (Rho-kinase) or 15% (RhoA) SDS-polyacrylamide gel electrophoresis (at room temperature over night). Rat brain extract served as a positive control. Proteins were transferred to a nitrocellulose membrane (Immobilon-P, Millipore, Inc.) using a Bio-Rad Mini-Protean III apparatus (2 hours at 4º C) in the presence of 25 mm Trizma Base, mm glycine and 20% methanol. The nitrocellulose membrane was then incubated with 5% skimmed milk in phosphate buffered saline (30 minutes, room temperature). After blocking, the membrane was incubated overnight (4 o C) with primary goat polyclonal antibody to the carboxy terminus of Rho-kinase (1:500 dilution, ROCK-2, Transduction Labs, Inc.) or primary monoclonal antibody to RhoA, (1:100 dilution, Transduction Labs, Inc.), and subsequently incubated for (2 hours at 4 o C) with an rabbit anti-mouse antibody (1:500 dilution, Transduction Labs, Inc.). Antibody-bound protein was visualized using an 125 I-protein A incubation (2 hours 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 BCA Protein assay (Pierce Biotechnology, Inc.) 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 Inc., Newport RI, USA. Cholesterol and phenylephrine were purchased from Sigma Chemical Co., St Louis, MO, USA. Data Analysis and Statistics Raw force responses were recorded digitally with POLYview data acquisition (Astro-Med, Inc., West Warwick, RI). Post-acquisition analysis included conversion of force (in grams) to stress (mn/mm 2 ). Force was converted to stress values (Force/cross-sectional area) by the following equation: 7

8 [(force (mg) ) / area] / (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 EC 50 s. Data were presented as mean ± SEM. 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 < 0.05 was considered to be significant. 8

9 Results Final Excepted Version R R1 Effect of castration and testosterone-treatment on the voltage-dependent erectile response. To examine the voltage-dependent erectile response, the major pelvic ganglion (MPG) was stimulated over a range of 1-5 volts, while mean arterial pressure (MAP) and corpus cavernosal pressure (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 voltagedependent increase in CCP/MAP, in accordance with previously published findings (9,21,37). Surgical castration of male rats (10 days post-surgery) resulted in a depressed erectile response to voltage stimulation of the major pelvic ganglion as compared to intact age-matched animals (Figure 1). Statistically significant depression in the erectile responses was seen in the castrate group at voltages greater than 3 (Figure 1B, left panel). 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 (Figure 1A and 1B, left panel). Castration and testosterone-treatment had no effect on MAP or CCP prior to MPG stimulation (Table 1). Effect of Y 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 upregualtion of RhoA/Rho-kinase signaling and could therefore reversed by inhibition of Rho-kinase activity. Injection of the selective Rho-kinase antagonist, Y (5 µl 20 nm) into the left corpus cavernosum sinuses resulted in an increase in CCP/MAP in all experimental groups without MPG stimulation (Figure 1A and 1B, right panel). We found that the intracavernosal injection of Y had a small but not significant depression in MAP in all experimental groups, while producing a significant 3 fold elevation in CCP in both castrate and testosterone-treated groups without MPG stimulation (Table 1). The doubling in CCP with Y 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 We next examined the effect of Y on the voltage-dependent increase in CCP/MAP. Y (5µl of 20 nm) was injected into the left corpus cavernosum and the increase in CCP pressure was allowed to plateau (5 minutes after injection). The subsequent CCP/MAP response to MPG stimulation 9

10 was assessed over the voltage range of 0-5 volts. The increase in CCP/MAP with MPG stimulation following administration of Y was elevated in all treatment groups (Figure 1B, right panel). Saline or vehicle injections produced no significant effect on MAP or CCP (data not shown). Administration of Y resulted in an improvement of the voltage-dependent erection of castrate group to levels that were not significantly different from the intact or testosterone-treated group responses (Figure 1B, right panel). α-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 α-adrenergic agonist phenylephrine (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 above 10 µm. Cavernosal strips from castrate animals displayed a monotonic response with a plateau in stress above 10 µm and a maximal stress response that was significantly larger than the intact and testosterone-treated responses (Figure 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 EC 50 values (Intact, ± µm; Testosterone-treated, ± µm; *Castrate, ± µm, *P <0.05) (Figure 2B). Effect of Y on α-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 The increasing concentrations of Y 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 pre-stimulation in corpora from castrate animals (Figure 3A). However, the sensitivity to Rho-kinase inhibition was not different between the experimental groups with average EC 50 values of: Intact, ± µm; Castrate, ± µm; Testosterone-treated, ± µm (Figure 3B). 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 α-adrenergic agonist PE. We sought to examine the impact of 10

11 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 Rhokinase in the cavernosal tissues (Figures 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 greater than 15 fold increase in the amount of RhoA detected by western blotting (Figure 4B). The mean optical density units for RhoA, normalized for the total protein levels were: Intact, 6.3 ± 1.0; *Castrate, 74.3 ± 8.1; *Testosterone-treated, 57.8 ± 10.5 (* values different from intact, P < 0.05, n = 4-9). A similar pattern of protein detection was seen with Rho-kinase. Castration resulted in a greater than 2 fold increase in the amount of Rho-kinase detected by western blotting (Figure 5B). The mean optical density units for Rho-kinase normalized for the total protein levels were: Intact, 16.0 ± 3.6; *Castrate, 27.5 ± 6.6; *Testosterone-treated, 24.9 ± 12.5 (* values different from intact, P < 0.05, n = 3-6). The 70 kda band seen in the RhoA and Rho-kinase blots were determined to be non-specific staining. 11

12 Discussion Final Excepted Version R R1 The findings reported from these studies demonstrate that castration decreased erectile response to ganglionic stimulation and increased constrictor responsiveness to phenylephrine 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 nitric oxide or its signaling pathway, little emphasis has been placed on other regulators of erectile function. We were particular 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. Intra-cavernosal injection of the Rho-kinase inhibitor, Y (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 (Figure 1). The suppressed erectile response was associated with an increased expression of both Rho-kinase and its upstream regulator RhoA (Figures 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 nitric oxide release (26-28). We found, in these studies, 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 to intact age-matched controls (Figure 1). The in vitro constrictor response to the α-adrenergic agonist phenylephrine was elevated in corpus cavernosal strips from the castrate animals (Figure 2). This response differs from that reported by Alcorn and co-workers who examined the contractile response to 100 µm norepinephrine and found no difference between intact, castrate and testosterone-treated animals (1). 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 phenylephrine which may reflect the receptor desensitization to this high agonist concentration. 12

13 Using a rat castrate model similar to the one employed in these studies Reilly and co-workers found that a portion of the depressed erectile response associated with castration was attributed to an increased α- adrenergic responsiveness (27). 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 α1- adrenergic agonist, phenylephrine, was shifted to the right in castrated animals (Figure 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 cites studies that demonstrated changes in receptor populations with erectile disorders and that some of the most recently identified α1 receptor subtypes have different affinities for selective antagonists (2). Traish and co-workers reported that castration reduced the expression of α1-adrenergic receptor in cavernosal tissue, and whose reduction was prevented or reversed by testosterone-replacement (34). Our data is consistent with a possible change in receptor population or subtype as we observed a change in maximal response and a shift in the calculated EC 50 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 α-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 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). 13

14 This may result in not only restoration of a NO-mediated relaxation via increased Ca 2+ sequestration and decreased myosin light chain kinase activity but also through the inhibition of Rho-kinase and maintained myosin phosphatase activity. The calculated EC 50 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 (36) for rabbit permeabilized corpora cavernosal tissues while our values were slightly lower or equal to those reported by Rees in intact tissues of human (2.2 µm) and rabbit (1.0 µm) cavernosum (24). In light of the similar EC 50 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 Rhokinase 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 ED 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 guanine nucleotide dissociation inhibitor (GDI) which 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 significant as most previous studies have examined altered NO relaxation mechanisms and not mechanisms associated with maintenance of smooth muscle tone. 14

15 Acknowledgements Final Excepted Version R R1 This work was supported by grants from the National Institutes of Health (NIH DK59467), American Health Assistance, National Heart Foundation H ) and the MCGRI grants award program awarded to C. J. Wingard. Thanks are extended to Heather Branam and Paula Jackson for technical assistance in performing the experiments. Andre Holmes was a participant in the MCG SEEP Research Program. Y was a gracious gift from the Mitsubishi Pharma of Osaka, Japan. Reference List 1. Alcorn, J. F., J. R. Toepfer, and R. E. Leipheimer. The effects of castration on relaxation of rat corpus cavernosum smooth muscle in vitro. J Urol 161: , Andersson, K. E. Pharmacology of penile erection. Pharmacol Rev 53: , Arver, S., A. S. Dobs, A. W. Meikle, R. P. Allen, S. W. Sanders, and N. A. Mazer. Improvement of sexual function in testosterone deficient men treated for 1 year with a permeation enhanced testosterone transdermal system. J Urol 155: , Baba, K., M. Yajima, S. Carrier, D. M. Morgan, L. Nunes, T. F. Lue, and T. Iwamoto. Delayed testosterone replacement restores nitric oxide synthase-containing nerve fibres and the erectile response in rat penis. BJU Int 85: , Bivalacqua, T. J., M. Rajasekaran, H. C. Champion, R. Wang, S. C. Sikka, P. J. Kadowitz, and W. J. Hellstrom. The influence of castration on pharmacologically induced penile erection in the cat. J Androl 19: , Chamness, S. L., D. D. Ricker, J. K. Crone, C. L. Dembeck, M. P. Maguire, A. L. Burnett, and T. S. Chang. The effect of androgen on nitric oxide synthase in the male reproductive tract of the rat. Fertil Steril 63: , Chitaley, K., R. C. Webb, A. M. Dorrance, and T. M. Mills. Decreased penile erection in DOCA-salt and stroke prone-spontaneously hypertensive rats. Int J Impot Res 13 Suppl 5: S16- S20,

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17 17. Mills, T. M., K. Chitaley, R. W. Lewis, and R. C. Webb. Nitric oxide inhibits RhoA/Rho-kinase signaling to cause penile erection. Eur J Pharmacol 439: , Mills, T. M., K. Chitaley, C. J. Wingard, R. W. Lewis, and R. C. Webb. Effect of Rho-kinase inhibition on vasoconstriction in the penile circulation. J Appl Physiol 91: , Mills, T. M., R. Lewis, C. J. Wingard, K. Chitaley, and R. C. Webb. Inhibition of tonic contraction-a novel way to approach erectile dysfunction? J Androl 23, S5-S Mills, T. M. and R. W. Lewis. The Role of Andorgens in the Erectile Response: A 1999 Perspective. Mol Urol 3: 75-86, Mills, T. M., D. M. Pollock, R. W. Lewis, H. S. Branam, and C. J. Wingard. Endothelin-1- induced vasoconstriction is inhibited during erection in rats. Am J Physiol Regul Integr Comp Physiol 281: R476-R483, Mills, T. M., C. M. Reilly, and R. W. Lewis. Androgens and penile erection: a review. J Androl 17: , Mills, T. M., V. S. Stopper, and C. M. Reilly. Sites of androgenic regulation of cavernosal blood pressure during penile erection in the rat. Int J Impot Res 8: 29-34, Rees, R. W., D. J. Ralph, M. Royle, S. Moncada, and S. Cellek. Y-27632, an inhibitor of Rhokinase, antagonizes noradrenergic contractions in the rabbit and human penile corpus cavernosum. Br J Pharmacol 133: , Rees, R. W., T. Ziessen, D. J. Ralph, P. Kell, S. Moncada, and S. Cellek. Human and rabbit cavernosal smooth muscle cells express Rho-kinase. Int J Impot Res 14: 1-7, Reilly, C. M., R. W. Lewis, V. S. Stopper, and T. M. Mills. Androgenic maintenance of the rat erectile response via a non-nitric-oxide-dependent pathway. J Androl 18: , Reilly, C. M., V. S. Stopper, and T. M. Mills. Androgens modulate the alpha-adrenergic responsiveness of vascular smooth muscle in the corpus cavernosum. J Androl 18: 26-31,

18 28. Reilly, C. M., P. Zamorano, V. S. Stopper, and T. M. Mills. Androgenic regulation of NO availability in rat penile erection [see comments]. J Androl 18: , Sauzeau, V., H. Le Jeune, C. Cario-Toumaniantz, A. Smolenski, S. M. Lohmann, J. Bertoglio, P. Chardin, P. Pacaud, and G. Loirand. Cyclic GMP-dependent protein kinase signaling pathway inhibits RhoA-induced Ca2+ sensitization of contraction in vascular smooth muscle. J Biol Chem 275: , Schirar, A., C. Bonnefond, C. Meusnier, and E. Devinoy. Androgens modulate nitric oxide synthase messenger ribonucleic acid expression in neurons of the major pelvic ganglion in the rat. Endocrinology 138: , Somlyo, A. P. and A. V. Somlyo. Signal transduction and regulation in smooth muscle. Nature 372: , Somlyo, A. P. and A. V. Somlyo. Signal transduction by G-proteins, rho-kinase and protein phosphatase to smooth muscle and non-muscle myosin II. J Physiol (Lond) 522 Pt 2: , Tokumitsu, M., S. Kaneko, A. Numata, N. Taniguchi, and S. Yachiku. Nocturnal penile tumescence (NPT) and its mechanism. Nippon Rinsho 60 Suppl 6: 76-80, Traish, A. M., K. Park, V. Dhir, N. N. Kim, R. B. Moreland, and I. Goldstein. Effects of castration and androgen replacement on erectile function in a rabbit model. Endocrinology 140: , Uehata, M., T. Ishizaki, H. Satoh, T. Ono, T. Kawahara, T. Morishita, H. Tamakawa, K. Yamagami, J. Inui, M. Maekawa, and S. Narumiya. Calcium sensitization of smooth muscle mediated by a Rho-associated protein kinase in hypertension. Nature 389: , Wang, H., M. Eto, W. D. Steers, A. P. Somlyo, and A. V Somlyo. RhoA Mediated Ca2+sensitization in Erectile Fucntion. J Bio Chem 277(34), ,

19 37. Wingard, C. J., R. Lewis, and T. M. Mills. Erection and NO Overide the Vasoconstrictive Effect of α-adrenergic Stimulation in the Rat Penile Vasculature. Int J Impot Res 13: 1-9, Zvara, P., R. Sioufi, H. M. Schipper, L. R. Begin, and G. B. Brock. Nitric oxide mediated erectile activity is a testosterone dependent event: a rat erection model. Int J ImpotRes 7: ,

20 FIGURE 1 Voltage-dependent erectile response of Intact, Castrate, & Testosterone-treated rats before and after Rho-kinase inhibition. Panel A: Representative traces of the erectile response (CCP/MAP) to graded voltages applied to the major pelvic ganglion before and after intracavernosal injection of the Rho-kinase inhibitor, Y Panel B: Erectile responses for each applied voltage (based on the average value for 2 minutes of stimulation at a given voltage) Mean responses ± 1 SEM are reported. Left panel: The erectile responses from animals before administration of 5 µl 20 nm Y Right panel: The erectile responses from the same animals 5 minutes after administration of Y n = 5-6 animals in experimental group, * statistical significance from Intact values P < FIGURE 2 Stress-generation responses of isolated cavernosal strips to increasing concentration of the α-adrenergic agonist phenylephrine. Panel A: Increases in phenylephrine concentration were associated with increased stress generation in all experimental groups over the µm range. Castration was associated with a significant right-ward shift of concentration-response relationship and testosterone treatment returned the concentration-response relationship to that of the intact (Panel B). n = 4-6, * statistical significance from Intact values P < Labeled EC 50 values are based on mean data. FIGURE 3 Relaxation of isolated cavernosal strips to graded inhibition of Rho-kinase by Y Panel A: Increases in Y concentration were associated with reductions in stress induced by 10 µm PE in all experimental groups over the µm range. Castration and testosterone-treatment was not associated with any significant shift in the concentration-response relationship (Panel B). n = 4-6, * statistical significance from Intact values P < Labeled EC 50 values are based on mean data. FIGURE 4 Immunodetection of RhoA in cavernosal tissue from Intact, Castrate, and Testosteronetreated animals. Panel A: representative western blots for RhoA from tissue homogenates of isolated cavernosal strips. In each lane 100 µg of total protein was loaded. Panel B: graph presents quantitation of fold change in optical density of 23 kd band which corresponded to RhoA. n = 4-9 animals, *indicates statistical significance from intact levels at P <

21 FIGURE 5 Immunodetection of Rho-kinase in cavernosal tissue from Intact, Castrate, and Testosterone-treated animals. Panel A: representative western blots for Rho-kinase from tissue homogenates of isolated cavernosal strips. In each lane 100 µg of total protein was loaded. Panel B: graph presents quantitation of fold change in optical density of 180 kd band which corresponds to Rhokinase. N= 3-6 animals, *indicates statistical significance from Intact levels at P <

22 Table 1 Mean arterial (MAP), corporal cavernosal (CCP) pressures in response to CCP injection of 5 µl of 20 nm Y prior to MPG stimulation. MAP CCP Treatment Before Y After Y Before Y After Y Intact ± ± ± ± 4.0 Castrate 98.5 ± ± ± ± 7.1* Testosteronetreated ± ± ± ± 7.0* Reported are Mean values ± SEM. indicates near statistical significance at P = 0.06 while * Indicates statistical significance P < 0.05 from Before Y value. n = 4. 22

23 A CCP/MAP Break (5min) Intact Cast Testo nm Y Time (min) B 1.0 Before Y After Y Intact Cast Testo 0.8 CCP/MAP * * * Y Volts Volts 23

24 A Active Stress (mn/mm 2 ) Intact Cast Testo * * * * B [PE] (µm) Normalized Stress (% maximal response) Intact Cast Testo * * * EC 50 = 0.73 µm EC 50 = 0.21 µm EC 50 = 0.30 µm [PE] (µm) 24

25 A Active Stress (mn/mm 2 ) * * * Final Excepted Version R R1 Intact Cast Testo [Y-27632] (µm) B 100 Normalized Relaxation (% of 10 µm PE) Intact EC 50 = 0.91 µm Cast EC 50 = 1.26 µm Testo EC 50 = 0.96 µm [Y-27632] (µm) 25

26 A Intact Castrate Testo Rat Brain 66kd 20kd RhoA B Fold Change in Optical Density * * Intact Castrate Testo Treatment 26

27 A 220Kd Intact Castrate Testo Rat Brain Rho-kinase 66kd B Fold Change in Optical Density * * Intact Castrate Testo Treatment 27