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CELLULAR AND MOLECULAR
Department of Physiology, Medical College of Georgia, Augusta, Georgia
Received for publication
March 8, 2005
Accepted
June 14, 2005.
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Abstract |
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, 2003). The main contractile factor is thought to be noradrenaline released from sympathetic nerve endings, acting on postjunctional 
1-adrenoceptors (Andersson, 2001, 2003). The mechanism of noradrenergic contraction is based on the activation of subcellular signaling systems that mobilize Ca2+ from extracellular and intracellular stores, resulting in an increased intracellular Ca2+ concentration ([Ca2+]i) (Himpens et al., 1995). The elevation of [Ca2+]i causes calmodulin-mediated activation of myosin light chain (MLC) kinase, MLC phosphorylation, and cross-bridge cycling, which results in contraction. On the other hand, a decrease in [Ca2+]i permits dephosphorylation of MLC by MLC phosphatase, thus, causing relaxation (Somlyo and Somlyo, 2003).
In addition, distinct regulatory mechanisms brought about by activation of heterotrimeric G protein-coupled receptors are known to elevate the degree of contractile force without further increases in [Ca2+]i, a process termed Ca2+ sensitization. One of these mechanisms involves RhoA, a small, monomeric G protein that activates Rho-kinase, which in turn phosphorylates the regulatory subunit of MLC phosphatase and leads to sensitization of myofilaments to Ca2+ (Riento and Ridley, 2003; Somlyo and Somlyo, 2003). Rho-kinase is expressed in human, rabbit, and rat cavernosal smooth muscle (Mills et al., 2001; Rees et al., 2002; Wang et al., 2002). Using an in vivo rat model, we have demonstrated that a Rho-kinase inhibitor, Y-27632, caused a dramatic increase in intracavernosal pressure by a mechanism independent of nitric oxide (NO), with minimal effects on systemic blood pressure (Chitaley et al., 2001). In addition, this inhibitor has been shown to antagonize noradrenergic contractions in human and rabbit penile tissue (Rees et al., 2002; Wang et al., 2002; Takahashi et al., 2003). These findings collectively indicate that the RhoA/Rho-kinase pathway is present and active in the resting (contracted) state of the corpus cavernosum.
A pivotal role for NO in the relaxation of the corpus cavernosum and vasculature is widely accepted (Andersson and Wagner, 1995). The endothelium and nerve fibers innervating the cavernosal tissue may be the source of NO, which is released after stimulation of neuronal and endothelial NO synthase (eNOS), respectively. Chitaley et al. (2001) have reported that cavernosal relaxations induced by Y-27632 were not affected by treatment with inhibitors of either NO synthase or soluble guanylyl cyclase. In contrast, recent studies reported that stimulation of the cGMP-dependent protein kinase leads to phosphorylation of RhoA and its inhibition. Therefore, inhibition of RhoA and its downstream target Rho-kinase by NO would contribute to the vasodilator action of NO (Sauzeau et al., 2000; Sandu et al., 2001; Chitaley and Webb, 2002).
Because cavernosal smooth muscle is kept contracted by basal noradrenergic activity in the detumescent state, we were interested in investigating the in vitro and in vivo effects of the novel Rho-kinase inhibitor H-1152 (Sasaki et al., 2002) in the penile tissue. Moreover, multiple mechanisms were recently described to link the Rho-kinase pathway and NO-generating system; therefore, we decided to reexamine the role of endogenous NO in the modulation of the mechanisms underlying Ca2+ sensitization in the corpus cavernosum. Furthermore, previous studies demonstrated that the regulator of G protein signaling (RGS) domain is responsible for the physical association with the G subunit of GTPase and acts to regulate G protein-coupled receptor signaling (Ross and Wilkie, 2000; Sah et al., 2000; Schmidt and Hall, 2002). Therefore, to further explore the signal transduction pathway for the increases in Ca2+ sensitivity, we investigated the mRNA expression of the RGS-containing Rho guanine nucleotide exchange factors (RhoGEFs) in the corpus cavernosum.
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Materials and Methods |
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Drugs and Solutions. Krebs' solution of the following composition was used: 130 mM NaCl, 14.9 mM NaHCO3, 5.5 mM dextrose, 4.7 mM KCl, 1.18 mM KH2PO4, 1.17 mM MgSO4·7H2O, and 1.6 mM CaCl2·2H2O. Acetylcholine, bretylium tosylate, N
-nitro-L-arginine methyl ester (L-NAME), 1H-[1,2,4]oxadiazolo [4,3,-a]quinoxalin-1-one (ODQ), phenylephrine, prazosin, sodium nitroprusside, and tetrodotoxin were purchased from Sigma Chemical Co. (St. Louis, MO). Pentobarbital sodium was obtained from the university pharmacy. H-1152 and Y-27632 were acquired from Calbiochem (San Diego, CA). TRIzol and superscript II kit were purchased from Invitrogen (Carlsbad, CA). All other reagents used were of analytical grade. Stock solutions were prepared in deionized water and stored in aliquots at –20°C; dilutions were made up immediately before use. ODQ was prepared in dimethyl sulfoxide. Solutions were stored at –20°C and further diluted in deionized water just before use.
Functional Studies in Cavernosal Strips. The animals were anesthetized with pentobarbital sodium (40 mg/kg i.p.), killed by decapitation, and exsanguinated. The penis was excised, transferred into ice-cold Krebs' solution, and surgically dissected to remove the tunica albuginea. One crural strip preparation (1 x 1 x 10 mm) was obtained from each corpus cavernosum. Cavernosal strips were mounted in 4-ml myograph chambers (Danish Myo Technology, Aarhus, Denmark) containing Krebs' solution at 37°C continuously bubbled with a mixture of 95% O2 and 5% CO2. The tissues were stretched to a resting force of 2 mN and allowed to equilibrate for 60 min. Changes in isometric force were recorded using a PowerLab/8SP data acquisition system (Chart software, version 5.0; ADInstruments, Colorado Springs, CO). To verify the contractile ability of the preparations, a high K+ solution (80 mM) was added to the organ baths at the end of the equilibration period. Cumulative concentration-response curves to H-1152 (0.001–3 µM) and Y-27632 (0.01–30 µM) were obtained in cavernosal strips contracted with phenylephrine (PE; 10 µM). Electrical field stimulation (EFS) was applied in muscles placed between platinum pin electrodes attached to a stimulus splitter unit (Stimu-Splitter II), which was connected to a Grass S88 stimulator (Astro-Med West Warwick, RI). EFS was conducted at 50 V, 1-ms pulse width and trains of stimuli lasting 10 s at varying frequencies (1–32 Hz).
Measurement of Intracavernosal Pressure. Rats were anesthetized with intramuscular ketamine (87 mg/kg) plus xylazine (13 mg/kg) and maintained on supplemental ketamine as needed. The right carotid artery was cannulated for continuous monitoring of mean arterial pressure (MAP), and a 30-gauge needle was inserted into the right corpus cavernosum to record intracavernosal pressure (ICP) simultaneously via Grass pressure transducers (Astro-Med). The abdominal cavity was opened, exposing the right cavernosal nerve at the dorsolateral aspect of the prostate. Stainless steel bipolar electrodes connected to a Grass SD9 stimulator (Astro-Med) were positioned on the cavernosal nerve. Electrical stimulation of the cavernous nerve (5-ms pulse width; 60 s; 5 V) at different frequencies (1, 2, 4, 8, and 16 Hz) was performed. All pressure data were collected for analysis using PowerLab/8SP data acquisition system.
Primer Design and RT-PCR. Rat homologues of PDZ-RhoGEF, LARG, and p115RhoGEF were identified by comparative genome analysis using publicly available rat, mouse, and human data. Primers were designed with Primer3 program based on the known mRNA sequences for each gene. To exclude the possible contamination of genomic DNA, care was taken to ensure that the two primers for one gene were located at different exons. The PDZ-RhoGEF primers were as follows: forward, 5'-GGGACCCTCTTCGAGAACGCCAAA-3'; and reverse, 5'-GGGCAGCCAC-TTGTCCTTGTCAGG-3'. LARG primers were forward, 5'-AGCCATG-CGCGCTGGAGTACAAAC-3'; and reverse, 5'-GCTCCAGGGGAATGAGGGGATGTC-3'. p115RhoGEF primers were forward, 5'-TCCGGACCAAGAGTGGGGACAAGA-3'; and reverse, 5'-TACCCAGGCTTCCCTTCCGGTCTG-3'. Glyceraldehyde-3-phosphate dehydrogenase primers were forward, TGCATCCTGCACCACCAACTGCTT; and reverse, ACAGCCTTGGCAGCACC-AGTGGAT.
Penile tissue was isolated, snap-frozen in liquid nitrogen, and stored at –80°C for subsequent analysis. Total RNA (4 µg/reaction) extracted from tissue segments with TRIzol reagent (Invitrogen) was used for the first-strand cDNA synthesis with superscript II kit (Invitrogen), according to manufacturer's specifications. cDNA equal to 0.04 µg of total RNA was used for each PCR reaction under the following conditions: 94°C for 2 min and 22 (for glyceraldehyde-3-phosphate dehydrogenase) or 30 (for RhoGEFs) cycles at 94°C for 30 s, 60°C for 30 s, and 72°C for 30 s, followed by 72°C for 7 min. The reaction products were analyzed by electrophoresis on agarose gel and the expected product was extracted and verified by direct DNA sequencing. The gel images were recorded by videocamera (Sony Video Camera Module Charge-Coupled Device; Sony, Tokyo, Japan), connected to an IBM AT computer (IBM, White Plains, NY) with a 512 x 512-pixel array imaging board with 256 gray levels.
Statistical Analysis. Experimental values of relaxation were calculated relative to the maximal changes from the contraction produced by PE in each tissue, which was taken as 100%. Calculations were based on the number of experiments performed in each individual. In vivo erectile responses were expressed as the ratio of 
ICP (mm Hg)/MAP (mm Hg) x 100, with ICP, the difference between ICP in the flaccid state and ICP during the plateau phase of the erectile response and MAP, the mean arterial pressure during the plateau phase of the erectile response. During detumescence, the time (D20) and the rate (D20) for ICP to decrease to 20% of maximal increase were determined (Mizusawa et al., 2001). Data are shown as the percentage of relaxation of n experiments, expressed as the mean ± S.E.M. Analysis of variance and Student's paired t test were used to evaluate the results. A p value less than 0.05 was considered to indicate significance. A program package was used for the statistical analysis of all data (Instat, ver. 3.0; GraphPad Software Inc., San Diego, CA).
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Results |
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Effect of L-NAME and ODQ on Relaxations Induced by H-1152 and Y-27632. Under our experimental conditions, the incubation of rat cavernosal strips with either L-NAME (100 µM), a nonselective inhibitor of NO synthase, or ODQ (10 µM), an inhibitor of soluble guanylyl cyclase, virtually abolished the endothelium-dependent relaxations mediated by acetylcholine (10 µM). It is interesting that acetylcholine relaxed precontracted rat corporeal tissue at the above-mentioned concentration by only 10 ± 2%, unlike in corpus cavernosum isolated from humans, rabbits, or mice, where this concentration has been shown to cause 40 to 70% relaxation. The addition of L-NAME (n = 5) or ODQ (n = 4) caused a discrete contractile effect (
5% increase) on the tone induced by PE (10 µM). As shown in Table 1, treatment of cavernosal strips with either L-NAME or ODQ did not significantly affect the relaxations evoked by H-1152 (0.001–3 µM) or Y-27632 (0.01–30 µM).
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Effect of Rho-Kinase Inhibition on PE- and EFS-Induced Contractions. Addition of PE (0.01–100 µM) to the bathing medium concentration dependently contracted the cavernosal smooth muscle strips with a pEC50 value of 6.27 ± 0.06 (n = 18), reaching a peak (166 ± 4% of KCl-induced contraction) at 30 µM. Addition of the Rho-kinase inhibitors H-1152 (0.1–1 µM) or Y-27632 (1–10 µM) significantly attenuated the contractions evoked by PE in a concentration-dependent manner. At their highest concentration, H-1152 and Y-27632 caused 9.7- and 11.5-fold rightward shifts in the curves to PE, respectively (n = 4; p < 0.01). The NO donor SNP (0.1–1 µM; n = 4) also inhibited the contractile activity evoked by PE in a concentration-dependent manner, culminating with a 6.7-fold shift to the right after treatment of the preparations with 1 µM SNP. As shown in Fig. 2, H-1152 (0.1 µM), Y-27632 (1 µM), and SNP (0.1 µM) caused similar rightward shifts in the curves to PE of 2.8-, 2.9- and 2.4-fold, respectively (n = 4). However, the coincubation of H-1152 (0.1 µM) or Y-27632 (1 µM) with SNP (0.1 µM) resulted in significantly higher shifts in the curves to PE, compared with their actions individually, as represented by the 9.9- (H-1152 plus SNP) and 7.5-fold (Y-27632 plus SNP) displacement to the right (Fig. 2; p < 0.01).
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1-adrenergic antagonist prazosin (1 µM; n = 5). In addition, electrically induced contractions were fully blocked by the noradrenergic nerve blocking agent bretylium tosylate (30 µM; n = 6), confirming that these responses are neuronal in origin and adrenergic in nature. Addition of H-1152 (0.1–1 µM), Y-27632 (1–10 µM), or SNP (0.1–1 µM) caused concentration-dependent reductions of EFS-induced contractions, with maximal inhibitions at their highest concentration of 42 ± 4, 44 ± 3, and 16 ± 3%, respectively, when the strips were stimulated at 32 Hz (n = 4, each). As shown in Fig. 3, each agent had little effect on the contractile activity elicited by EFS over the full range of the frequency-response curve when their lowest concentration was used (H-1152, 0.1 µM; Y-27632, 1 µM; and SNP, 0.1 µM). However, the combination of either Rho-kinase inhibitor with SNP produced a significant inhibition of the electrically evoked contractions compared with the controls (Fig. 3; p < 0.01; n = 4).
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Intraperitoneal administration of H-1152 (100 nmol/kg; n = 4) did not considerably affect the ICP/MAP ratio in the absence of electrical stimulation of the cavernous nerve (0.07 ± 0.01 in control conditions, 0.08 ± 0.01 after 5 min, and 0.09 ± 0.01 after 15 min of H-1152 administration), despite a 12 ± 3% decrease in MAP. However, H-1152 significantly enhanced the responses evoked by electrical stimulation (Fig. 4). The enhancement by H-1152 was revealed both in the magnitude of the erectile responses as well as in the poststimulation period (Table 2). The potentiation of electrical stimulation-induced increases in ICP was also demonstrated by the measurement of the area under the curve (AUC) parameter in vehicle- and H-1152-treated animals. Indeed, AUC was increased by 42 ± 7% after 15 min of H-1152 administration (p < 0.01), whereas no significant change was noted in vehicle-treated animals (n = 4). As summarized in Table 2, administration of H-1152 caused an increase in the time (D20) and a decrease in the rate (D20) for ICP to decrease to 20% of the peak response.
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Expression of RGS-Containing RhoGEFs. An RT-PCR assay was used to measure the mRNA expression of the RGS-containing RhoGEFs in the rat corpus cavernosum, according to a methodology developed in our laboratory (Ying et al., 2004). Complimentary DNAs prepared by reverse transcription of total RNA extracted from rat celiac arteries coding for PDZ-RhoGEF, p115RhoGEF, and LARG were detectable. As a representative example, Fig. 5 demonstrates the electrophoretic visualization of the polymerase chain reaction products obtained from the amplification of cDNAs. In all samples, the fragments exhibiting the expected sizes were amplified without any contamination from nonspecific products (210 bp for PDZ-RhoGEF, 226 bp for p115RhoGEF, and 237 bp for LARG).
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Discussion |
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; Rees et al., 2002; Wang et al., 2002; Takahashi et al., 2003) and in vivo (Chitaley et al., 2001) studies of erectile function have shown that the RhoA/Rho-kinase signaling pathway is implicated in the regulation of cavernosal smooth muscle tone because of the inhibition of MLC phosphatase activity, resulting in increased MLC phosphorylation and force in the penile tissue. First, we compared the relaxant responses of two Rho-kinase inhibitors, H-1152 and Y-27632, in the isolated rat corpus cavernosum, by applying either compound to strips precontracted with the 1-agonist phenylephrine. The contractions were concentration dependently inhibited by H-1152 and Y-27632, indicating that the Rho-kinase pathway is involved in the adrenergic contraction of the cavernosal tissue, supporting previous studies (Chitaley et al., 2001; Rees et al., 2002; Wang et al., 2002; Takahashi et al., 2003). The tonic force produced by phenylephrine in rat corpus cavernosum was more sensitive to H-1152 than Y-27632, because the former compound was approximately 10 times more potent than the later, rendering H-1152 as a more effective inhibitor of Rho-kinase.
Previous studies demonstrated that in vascular smooth muscle RhoA activation is accompanied by the membrane translocation of GTP-bound RhoA (Fujihara et al., 1997; Seasholtz et al., 1999; Gong et al., 2001). On the other hand, the NO signaling pathway exerts an inhibitory action on RhoA function via cGMP-dependent protein kinases, which phosphorylate RhoA, thus inhibiting its translocation to the membrane (Gohla et al., 2000; Sauzeau et al., 2000; Sandu et al., 2001; Sawada et al., 2001; Chitaley and Webb, 2002). In rat cavernosal strips, we found that neither L-NAME nor ODQ affected the relaxant responses evoked by H-1152 or Y-27632, corroborating previous observations that inhibition of NOS or soluble guanylyl cyclase does not influence relaxations mediated by Rho-kinase inhibitors (Chitaley et al., 2001). Nevertheless, although pharmacological inhibition of the NO/cGMP pathway failed to affect both H-1152- and Y-27632-evoked relaxations, the lines of evidence presented in this study suggest that attenuation of RhoA/Rho-kinase signaling in rat cavernosal smooth muscle represents part of the NO-induced inhibitory effects on contractile activity, as evidenced previously in vascular smooth muscle. First, relaxations evoked by acetylcholine at a concentration that markedly relaxes both human and rabbit cavernosal preparations (10 µM), caused only a small relaxant response in precontracted rat corporeal tissue, in agreement with previous studies (Hedlund et al., 1999; Claudino et al., 2004). Second, addition of either L-NAME or ODQ had little effect on the tone of the preparations, whereas these inhibitors have been shown to induce marked contractions in the corpus cavernosum from other species (Teixeira et al., 2004a,b). Although a positive NADPH diaphorase has been described in some parts of the sinusoidal endothelium of the rat penis (Keast, 1992), this has not been found in later investigations (Schirar et al., 1994; Dail et al., 1995; Hedlund et al., 1999). Bivalacqua et al. (2004) have demonstrated that the RhoA/Rho-kinase pathway down-regulates rat penile eNOS in diabetes and that inhibition of Rho-kinase led to a restoration of the erectile function, which was accompanied by an improvement of eNOS expression. Based on these observations, one could argue in favor of endothelium-derived NO as a key mediator of erectile function. Nevertheless, the erectile process is initiated by stimulation of NO release from nitrergic nerves, with a subsequent dilation of the penile arteries to permit an increase in blood flow to the penis (Andersson and Wagner, 1995). In response to stretch and possibly shear stress, the sinusoidal endothelium may release NO to help maintain the erectile state. In this previous study (Bivalacqua et al., 2004), the functional data presented were obtained in the intact animal by measuring intracavernosal pressure, thus representing an action of nerve-derived NO on the arteries and the sinuses (stretch). The authors did not test the responsiveness to acetylcholine in the isolated cavernosal tissue; therefore, a confusing issue remains with regard to the physiological basis for erection and the means to establish this basis experimentally. Together, our present observations as well as the previous studies aforementioned (Schirar et al., 1994; Dail et al., 1995; Hedlund et al., 1999; Claudino et al., 2004; Teixeira et al., 2004a,b) rule out a significant role for endothelium-derived NO in the erectile responses of rat corpus cavernosum, thus explaining the poor acetylcholine-evoked relaxations as well as the lack of effect of L-NAME and ODQ to increase tone and to inhibit the relaxant responses induced by Rho-kinase inhibitors.
To investigate the nature of the interaction between NO and Rho-kinase inhibitors in erectile tissue, we applied H-1152, Y-27632, or SNP to rat corpus cavernosum stimulated with phenylephrine or EFS, because contraction of the cavernosal smooth muscle is maintained by noradrenaline released from noradrenergic nerves of the penis as a result of 1-adrenoceptor activation (Cellek, 2000; Andersson, 2001; Moreland et al., 2001). Whereas either compound alone elicited a 3-fold rightward shift in the concentration-response curves to phenylephrine, coapplication of H-1152 plus SNP or Y-27632 plus SNP led to a synergistic inhibition of phenylephrine-induced contractions. Likewise, at concentrations that did not affect the transient contractions elicited by EFS, treatment of cavernosal strips with a combination of H-1152/Y-27632 with SNP caused significant attenuations of electrically induced contractions. These data indicate that increased NO and decreased Rho-kinase activity synergize to induce cavernosal smooth muscle relaxation in vitro (this study) as well as penile erection in vivo (Mills et al., 2002).
Previous studies from this laboratory suggest that vasoconstriction in the penile circulation may be regulated, in part, by the RhoA/Rho-kinase Ca2+ sensitization pathway (Chitaley et al., 2001; Mills et al., 2001). Indeed, injection of Y-27632 in the cavernous sinuses causes dose-dependent increases in intracavernous pressure, supporting a role for Rho-kinase in the maintenance of penile flaccidity (Chitaley et al., 2001). In the present investigation, we have evaluated the in vivo effects of H-1152 in the erectile responses evoked by stimulation of the cavernous nerve. The results presented herein demonstrate the relationship among electrical stimulation of the cavernous nerve, intracavernous pressure and mean arterial pressure, with measurements made before and after administration of H-1152. Most interesting is the demonstration that intraperitoneal injection of H-1152 significantly enhances both the magnitude and the duration of the erectile responses. Thus, these results confirm that the Rho-kinase pathway plays an important role in the maintenance of the vasoconstrictive state of the cavernosal vasculature.
The activity of Rho itself is regulated by distinct groups of proteins, such as RhoGEFs, which facilitate the activation of Rho by promoting the dissociation of GDP and subsequent binding of GTP (Ross and Wilkie, 2000; Sah et al., 2000; Schmidt and Hall, 2002). A group of RhoGEFs has recently been identified and characterized by a moiety resembling a regulator of RGS domain that binds the activated subunit of GTPase. This group of RhoGEF proteins currently comprises three members: LARG, p115RhoGEF, and PDZ-RhoGEF (Ross and Wilkie, 2000; Schmidt and Hall, 2002; Somlyo and Somlyo, 2003). Although p115RhoGEF is found predominantly in hematopoietic cells (Girkontaite et al., 2001), PDZ-RhoGEF and LARG seem to be more widely expressed (Fukuhara et al., 1999, 2000; Kourlas et al., 2000). We have developed a method for semiquantification of mRNA expression of the RGS domain-containing RhoGEFs in biological samples (Ying et al., 2004). To our knowledge, this is the first study that demonstrates the presence of RGS domain-containing RhoGEFs in corpus cavernosum, suggesting their potential role in the regulation of cavernosal smooth muscle tone.
In conclusion, our results showed that the compound H-1152 can completely and more potently reverse the Ca2+ sensitization process in the cavernosal tissue, both in vitro and in vivo. On this basis, Rho-kinase antagonism represents another potential line of therapy in the treatment of erectile dysfunction, especially in cases where either noradrenergic tone is elevated or nitrergic/endothelial-dependent responses are blunted. Most importantly, the design and synthesis of selective inhibitors of RGS domain-containing RhoGEFs should provide the framework for defining new physiological roles for these factors, not to mention the possibility of identifying novel agents to treat erectile disorders.
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Footnotes |
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ABBREVIATIONS: MLC, myosin light chain; Y-27632, (R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide; SNP, sodium nitroprusside; NO, nitric oxide; eNOS, endothelial nitric-oxide synthase; H-1152, (S)-(+)-2-methyl-1-[(4-methyl-5-isoquinolinyl)sulfonyl]homopiperazine; RGS, regulator of G-protein signaling; RhoGEF, Rho guanine nucleotide exchange factor; L-NAME, N-nitro-L-arginine methyl ester; ODQ, 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one; PE, phenylephrine; EFS, electrical field stimulation; MAP, mean arterial pressure; ICP, intracavernosal pressure; RT-PCR, reverse transcription-polymerase chain reaction; LARG, leukemia-associated Rho guanine nucleotide exchange factor; D, detumescence; AUC, area under the curve; ACh, acetylcholine; CC, corpus cavernosum; GPCR, G-protein-coupled receptors; bp, base pair(s).
Address correspondence to: Dr. Cleber E. Teixeira, Department of Physiology, Medical College of Georgia, 1120 15th St., CA-3101, Augusta, GA 30912-3000. E-mail: cteixeira{at}mail.mcg.edu
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