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From the Division of Urology, University of California San Diego Medical Center, San Diego, California, and the Urology Research Laboratory, Veterans Medical Research Foundation, La Jolla, California.
| Correspondence to: Dr Mahadevan Rajasekaran, c/o Miriam Seva, Division of Urology, UCSD Medical Center, 200 West Arbor Dr (8897), San Diego, CA 92103-8897 (e-mail: mseva{at}ucsd.edu). |
| Received for publication July 14, 2004; accepted for publication September 20, 2004. |
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Abstract |
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Key words: Muscle, smooth, drug effects, penis physiology, biological aging, penile erection
Besides the well-established noradrenergic contraction mechanisms in the penis, an additional mechanism involving increased sensitivity to ionic calcium has recently been proposed (Chitaley et al, 2001; Wang et al, 2002). This pathway involves RhoA, a small monomeric G-protein that activates Rho-kinase. Activated Rho-kinase phosphorylates the regulatory subunit of smooth-muscle myosin phosphatase (SMPP-IM). Inhibitory phosphorylation of SMPP-IM leads to sensitization of myofilaments to Ca2+ (Somlyo and Somlyo, 2000). An age-related increase in RhoA expression has been documented in rat vascular tissues and has been felt to be responsible for age-associated vascular disorders (Miao et al, 2001). A specific inhibitor of Rho-kinase, (+)-(R)-transB (1-amino-ethyl)-N-(4-pyridyl) cyclohexacarboxamide dihydrochloride monohydrate (Y-27632), has been shown to relax vascular and nonvascular smooth muscle, and intracavernosal injection of Y-27632 in rats has been shown to induce penile erection (Chitaley et al, 2001). We have recently demonstrated that hypertension, another vascular risk factor for ED, is associated with elevated penile RhoA levels and that inhibition of Rho-kinase activity with Y-27632 was beneficial in attenuating the decline in erectile function in hypertensive rats (Wilkes et al, 2004). In addition, Rho-kinase inhibition has been shown to be effective in reversing ED in castrated hypogonadal rats (Wingard et al, 2003). The impact of Rho-kinase inhibition in age-related ED has not been explored.
The present study was designed to monitor age-related alterations in male erectile function and specifically to evaluate the role of Rho-kinase pathway in the Brown-Norway (BN) rat, a sensitive animal model of reproductive aging. We also examined the hypotheses that antagonism of Rho-kinase activity attenuates the age-related decline in male erectile function and that a combination of Rho-kinase antagonism and phosphodiesterase-5 (PDE-5) inhibition has a synergistic effect in improving the erectile response in these aging animals.
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Materials and Methods |
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The major pelvic ganglion (MPG) was identified by dissecting the fibrous
capsule posterior to the intersection of the lateral lobes of the prostate and
the vas deferens with cotton tips. The MPG was stimulated with a voltage via 2
platinum electrodes delivered from a Grass S48 stimulator (Grass-Telefactor,
West Warwick, RI). Stimulus levels of 1.5, 3, and 4.5 V were used for
durations of 1 minute each; the rats were given a 5-minute rest period between
stimulations. CCP levels during stimulation were recorded directly into a
computerized data acquisition program.
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In this formula, P(t) denotes the pressure wave data as a
function of time and 
t is the time increments between data
points. T is the period of the pressure wave. The 
symbol is
used to denote that the pressure data is incremental and not continuous.
We employed 3 protocols to test our hypotheses.
Protocol 1— The impact of aging as well as the correlation of blood pressure to electrical field stimulation (EFS)-induced erectile response was evaluated in young and old rats by stimulating the MPG at 1.5, 3.0, and 4.5 V and recording AP and CCP as described above.
Protocol 2— To test the hypothesis that the specific Rho-kinase antagonist Y-27632 (Calbiochem, San Diego, Calif) attenuates the age-related decline in male erectile function, old animals were subjected to Protocol 1 followed by the intracavernosal administration of Y-27632 (50 nmol). After 10 to 15 minutes, Protocol 1 was repeated.
Protocol 3— To test the hypothesis that a combination of Rho-kinase antagonism and PDE-5 inhibition may have a synergistic effect in improving the erectile response in old animals, we subjected the old animals to Protocol 1 and then to the intracavernosal administration of the PDE-5 inhibitor zaprinast (Sigma Chemicals, St Louis, Mo) (100 µg) followed by Y-27632 (50 nmol). After 10 to 15 minutes, Protocol 1 was repeated.
All drug solutions were stored in a freezer in amber bottles; working solutions were prepared on a frequent basis and kept cold until injection time. In both old and young animals, the drugs were administered intracavernosally in small volumes (5 µL) when CCP was at a baseline value. The effect of a single injection agent on CCP was measured until CCP returned to the preinjection level. The next injection was made at least 10 to 15 minutes after CCP had returned to a stable baseline. An injection of 5 µL of saline vehicle had no significant sustained effect on CCP (Wilkes et al, 2004).
Statistical Analysis
Data were presented as mean ± standard error of the mean.
Statistical differences were determined by ANOVA followed by Newman-Keuls
multiple comparison test, where relevant, and by 2-tailed unpaired Student's
t test. A P value of less than .05 was considered
significant.
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Results |
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Our results showed a significant difference between old and young rats in ganglionic stimulation-induced CCP changes. A striking difference in maximum CCP value as well as in the ratio of CCP to MAP was noticed between the groups at all stimulation levels (the Table; Figure 2a and b). At 1.5 V, the young rats achieved about 34% higher CCP than the old group (41.07 ± 8.18 and 27.19 ± 6.60 in the young and old groups, respectively; P < .05). At 3 V, the mean CCP in the young group was 64.66 ± 5.08 when compared with 42.33 ± 5.74 cm H2O in old rats (the young group achieved 35% higher CCP at this stimulation than their older counterparts; P < .05). Stimulations at 4.5 V showed a more pronounced difference than the 3-volt stimulation. The mean CCP in young rats was 53% greater than that in old rats (the CCPs were 70.50 ± 7.10 and 33.20 ± 4.16 for the young and old groups, respectively; P < .05) (Figure 2a).
A similar trend was observed for the CCP/MAP index. The differences in CCP/MAP ratio between young and old animals were significant (P < .05) at all the indicated stimulation levels and ranged between 33% and 71% (Table; Figure 2b).
Efficacy of Rho-Kinase and PDE-5 Inhibition on Neurogenic Erectile Response in Aging Rats
The effect of Rho-kinase inhibition on EFS-induced erectile response in old
rats as measured by changes in CCP is illustrated in
Figure 3a and b. In old rats,
the impact of Rho-kinase inhibition following intracavernosal Y-27632 could be
seen immediately. A significant improvement in erectile response was observed
at all voltage levels of stimulation
(Figure 3a; Table). In Y-27632-injected old
rats, at 1.5 V, the CCP was 51.14 ± 7.87 and the mean CCP/MAP was 0.678
± 0.123 as compared with the predrug stimulation values of 27.19
± 6.61 and 0.227 ± 0.094 (P < .05). At 3 V, the
Y-27632-treated old rats achieved a CCP of 64.51 ± 1.23 and a CCP/MAP
of 0.762 ± 0.051 (P < .05 when compared with the predrug
stimulation values). At 4.5 V, the Y-27632-treated rats had a mean CCP of
65.58 ± 12.87 and a mean CCP/MAP of 0.852 ± 0.050 (P
< .05 when compared with their predrug stimulation values).
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A combination of Rho-kinase inhibition with Y-27632 and PDE-5 inhibition with zaprinast resulted in a synergistic improvement in neurogenic erectile response in old rats (Figure 3a and b). Representative tracings of CCP changes are shown in Figure 4. This combination produced a consistent 12% to 27% improvement in erectile response when compared with the Rho-kinase inhibition alone (Figure 3a and b; Table). The mean increase in CCP and the index of CCP/MAP were significant at all the levels of stimulation (P < .05).
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Evaluation of Systemic Arterial Pressure Changes
MAP in old rats ranged from 120 to 150 cm H2O, whereas young
rats had a MAP range of 110 to 140 cm H2O. Changes in MAP following
MPG stimulation are illustrated in Figure
5a. There was a voltage-dependent decrease in MAP in both groups
of animals; however, there was no significant difference in these changes
between the groups at any voltage (Figure
5a). Intracavernosal injection of Y-27632 and zaprinast resulted
in a marginal (20-25 cm H2O) drop in MAP in old rats (not
significant; Figure 5b).
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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The impact of aging on male sexual function has been the focus of several previous investigations (Garban et al, 1995a,b; Carrier et al, 1997; Champion et al, 1999; Dahiya et al, 1999; Rajasekaran et al, 2002; Bivalacqua et al, 2003). These studies suggest that age-related physiological changes in the penis, such as a decline in male hormone levels, decreased NO production, increased contractility of the smooth muscle of the penile corpora, a decreased relative percentage of smooth-muscle fibers, and increased collagen content, may contribute to development of ED in this population. While most of the previous studies on the role of aging on erectile function have focused on the NO-cGMP pathway, little emphasis has been placed on other regulators of erectile function. We were particularly interested in exploring the contribution of a novel calcium-sensitizing RhoA/Rho-kinase pathway to the aging-related decline in erectile function.
The RhoA signaling pathway, through activation of Rho-kinase leading to inhibition of myosin phosphatase resulting in an increase in myosin light chain (MLC) phosphorylation and force in smooth muscle, has been recognized to play a role in several vascular disorders (Chrissobolis and Sobey, 2001; Mukai et al, 2001). Inhibiting the RhoA/Rho-kinase pathway has been shown to cause erection in rats (Chitaley et al, 2001; Mills et al, 2001). Y-27632 has been shown to selectively inhibit the contractile activity associated with RhoA/Rho-kinase without stimulating vasorelaxation directly (Mills et al, 2002). NO has recently been shown to inhibit RhoA-mediated vasoconstriction and produce an erectile response in rats, possibly through inhibition of RhoA migration into the smooth muscle (Mills et al, 2002). Lower bio-availability of NO in the aging penis would lead to a reduction in RhoA migration to the membrane as well as cGMP-mediated Rho-kinase inactivation. The net result would be an increased penile smooth-muscle tone, which may be responsible for the impaired erectile response seen in the aging rats. Inhibition of the RhoA/Rho-kinase pathway has been shown to improve erectile function in rat models associated with other ED risk factors such as hypertension and hypogonadism. We have recently demonstrated that inhibition of Rho-kinase activity with Y-27632 was beneficial in attenuating the decline in erectile function in hypertensive rats (Wilkes et al, 2004). Wingard et al (2003) have shown that Rho-kinase inhibition was effective in reversing ED in castrated hypogonadal rats. An age-related increase in RhoA expression has been recently documented in rat vascular tissues, and this has been implicated in age-associated vascular disorders (Miao et al, 2001). Our results in the present study confirm these observations on the role of the Rho-kinase pathway in male erectile function and further suggest that Rho-kinase inhibitors could be of benefit in the treatment of age-related ED.
As aging is known to be associated with alterations in the NO-cGMP pathway as well as the Rho-kinase pathway (Garban et al, 1995b; Carrier et al, 1997; Miao et al, 2001; Rajasekaran et al, 2002), we extended our studies to examine whether a combination of PDE-5 inhibition, which would prevent cGMP degradation, and Rho-kinase antagonism, which would reduce the penile smooth-muscle tone, could result in a synergistic improvement of erectile response in aging BN rats. Furthermore, recent evidence suggests interplay between the NO-cGMP-cAMP and RhoA/Rho-kinase pathways (Kim et al, 2000; Abdel-Latif, 2001; Sauzeau et al, 2000; Sakai et al, 2003). Sakai et al (2003) reported that accumulation of cAMP in cells inhibited the RhoA activation in tracheal smooth muscle. Using vascular smooth-muscle cells, Sauzeau et al (2000) demonstrated that cGMP inhibited RhoA-dependent calcium sensitization. This inactivation of the RhoA/Rho-kinase pathway has been proposed to be mediated via cyclic nucleotide-dependent protein kinases through phosphorylation of RhoA (Sauzeau et al, 2000; Abdel-Latif, 2001). These observations suggest that interactions between cyclic nucleotide signaling systems and the Rho-kinase pathways may be responsible for the observed drug synergism between zaprinast and Y-27632. These results further suggest that this combination warrants exploration for its potential to improve the erectile response in severe cases of ED, where a modest 41% improvement has been reported with PDE-5 inhibition alone (Jarow et al, 1999). In addition, Rho-kinase inhibition may be explored as an alternate form of therapy for patients to whom PDE-5 inhibitors cannot be administered; for example, those on antihypertensive medications such as nitrites. This novel experimental drug Y-27632 has a significant potential to evolve as an alternative medication to oral medications, such as Viagra, for a distinct segment of the large, aging-ED patient population.
In conclusion, the BN rat strain may serve as an ideal animal model for the evaluation of age-associated ED in humans. Rho-kinase inhibition may be a novel therapeutic target in age-related male ED. Combination therapy with PDE-5 and Rho-kinase inhibition also merits therapeutic exploration.
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Acknowledgments |
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Footnotes |
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