Endothelium‐Independent Relaxant Effect of Rubus Coreanus Extracts in Corpus Cavernosum Smooth Muscle

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Endothelium-Independent Relaxant Effect of Rubus Coreanus Extracts in Corpus Cavernosum Smooth Muscle Jun Ho Lee, MD,* Mee Ree Chae, MSc,†‡ Hyun Hwan Sung, MD,†‡ Mikyeong Ko, MSc,†‡ Su Jeong Kang, MSc,†‡ and Sung Won Lee, MD, PhD†‡ *Department of Urology, National Police Hospital, Seoul, Korea; †Genitourinary Disease Oriented Translational Research, Seoul, Korea; ‡Department of Urology, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea DOI: 10.1111/jsm.12183

ABSTRACT

Introduction. Rubus coreanus is a perennial shrub native to the southern part of the Korean peninsula. Although it is known that R. coreanus has a dose-dependent relaxation effect on rabbit corpus cavernosum (CC), the exact mechanism of action by which R. coreanus work is not fully known. Aims. To elucidate the direct effects of unripe R. coreanus extract (RCE) on CC smooth muscle cells. Methods. Dried unripe R. coreanus fruits were pulverized and extracted with 95% ethanol. Isolated rabbit CC strips were mounted in an organ-bath system, and the effects of RCE were evaluated. To estimate [Ca2+]i, we used a Fura-2 fluorescent technique. Main Outcome Measures. The effects of unripe RCE on ion channels and the intracellular Ca2+ concentration ([Ca2+]i) of CC. Results. RCE effectively relaxed phenylephrine (PE)-induced tone in rabbit CC, and removal of the endothelium did not completely abolish the relaxation effect of RCE. Tetraethylammonium (1 mM) did not inhibit RCE-induced relaxation in strips precontracted by PE in the organ bath. However, CaCl2-induced constriction of CC strips, bathed in Ca2+-free buffer and primed with PE, was abolished by RCE. In addition, RCE decreased basal [Ca2+]i in corporal smooth muscle cells. The increases of [Ca2+]i evoked by 60 mM K+-containing solution in A7r5 cells were suppressed by RCE, and RCE relaxed KCl-induced tone in endothelium-free CC, which indicated that RCE blocked the voltage-dependent Ca2+ channels (VDCCs). RCE decreased basal [Ca2+]i and the [Arg8]-vasopressin-induced [Ca2+]i increases in A7r5 cells, and RCE inhibited the contraction of endothelium-free CC induced by PE in Ca2+-free solution, which suggested that RCE might act as a modulator of corporal smooth muscle cell tone by inhibiting Ca2+ release from sarcoplasmic reticulum. Conclusion. RCE acts through endothelium-independent and endothelium-dependent pathways to relax CC. RCE may inhibit VDCCs and Ca2+ release from sarcoplasmic reticulum. Lee JH, Chae MR, Sung HH, Ko M, Kang SJ, and Lee SW. Endothelium-independent relaxant effect of Rubus coreanus extracts in corpus cavernosum smooth muscle. J Sex Med 2013;10:1720–1729. Key Words. Rubus Coreanus; Erectile Dysfunction; Calcium Channels; Corpus Cavernosum Smooth Muscle

Introduction

E

rectile dysfunction (ED) is a common condition, with a reported prevalence of 52% in men aged 40–70 years [1,2]. It can impact the patient’s quality of life by impairing interpersonal relationships, interfering with their sexual life, causing problems with partners, and increasing mental stress [3].

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The introduction of oral phosphodiesterase type 5 (PDE5) inhibitors has revolutionized the field of sexual medicine. The advent of these medications has made treatment of ED easy for both the physicians who prescribe these drugs and the patients who take them. However, some problems remain to be solved [4]. There are clear contraindications for the use of PDE5 inhibitors, especially in patients on medications containing nitrates, and © 2013 International Society for Sexual Medicine

Effect of R. Coreanus Extracts in Corpus Cavernosum PDE5 inhibitors are associated with several side effects, such as headache, facial flushing, dizziness, myalgia, and dyspepsia [5]. These side effects and contraindications may result in drop-out rates of between 30 and 50% in long-term use of PDE5 inhibitors [6]. All these limitations in the use of PDE5 inhibitors have prompted the search for new treatment modalities. The use of natural products obtained from traditional herbs is appealing because their safety has, to some degree, been proved by their being widely used over a long period of time. Rubus coreanus is a perennial shrub native to the southern part of the Korean peninsula. The dried unripe fruit of R. coreanus has been used to improve ED in traditional Korean medicine [7]. R. coreanus has a dose-dependent relaxation effect on rabbit corpus cavernosum (CC) [8], and a herbal formulation including R. coreanus enhances intracavernous pressure and nitric oxide-cyclic guanosine monophosphate (NO-cGMP) activity in penile tissues of spontaneously hypertensive male rats [9]. In the experiment for elucidating the mechanism of R. coreanus [7], R. coreanus evoked relaxation was not completely inhibited by (1H[1,2,4] oxidiazolo [4,3-a] quinoxalin-1-one) (ODQ) or N (G)-nitro-L-arginine methyl ester (L-NAME). These data indicate that R. coreanusinduced relaxation has another pathway that is not dependent of the endothelium. We focused on the effects R. coreanus on ion channels and the intracellular Ca2+ level ([Ca2+]i), which are important in regulating smooth muscle tone. Thus, in the present study, we investigated aforementioned issues using organ-bath studies and measuring of intracellular Ca2+. Aims

The aim of our study is to elucidate the direct effects of unripe R. coreanus extract (RCE) on CC smooth muscle cells. Methods

Plant Materials The unripe fruits of R. coreanus Miquel (Rosaceae) were collected from the Gochang region (Jeonbuk, Korea) in July 2009 and identified and authenticated by Dr. Chul Young Kim of the KIST Gangneung Institute. A voucher specimen (accession number RC-1001) has been deposited at the Natural Products Research Center of KIST Gang-

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neung Institute in Gangneung, Korea. The shadedried unripe R. coreanus fruits were pulverized and extracted three times with 95% ethanol. The extract was preserved with drying using a SpeedVac concentrator (Savant, Farmingdale, NY, USA) and stored at 4°C until use. The yield of dried extract from starting dried plant material was approximately 12.0%.

Chemicals All drugs and chemicals were purchased from Sigma Chemical Company (St. Louis, MO, USA) except udenafil. Udenafil (DA-8159, a PDE5 inhibitor) was provided by Dong-A Pharmaceutical Company (Seoul, South Korea). Udenafil and RCE were dissolved in dimethyl sulfoxide (DMSO) and freshly diluted in bath solution immediately before use. The final concentration of DMSO did not exceed 0.1%. All other drugs were prepared in distilled water. Corporal Tissue Strip Preparation All animal experiments were conducted with the approval of the Institute for Animal Care and Use Committee of the Samsung Medical Center (Seoul, South Korea). Sexually mature male New Zealand white rabbits (2.5 ⫾ 0.3 kg) were killed by air embolism into the ear vein. Subsequently, the entire penis was surgically excised and cleaned by removing the corpus spongiosum and urethra. The corporal tissue was then carefully dissected from the surrounding tunica. Three to four corporal strips of approximately equal size (2 ¥ 2 ¥ 10 mm) were obtained from each penis and were prepared for organ-bath studies separately. Each corporal strip was tied with silk in one organ chamber, with one end fixed to a tissue holder and the other secured to a force transducer. The latter was connected to an appropriately calibrated four-channel polygraph (PowerLab; ADInstruments, Sydney, Australia) in which the transducer output was recorded. Corporal strips were maintained in the organ baths during the study with Krebs solution at 37°C by a thermoregulated water circuit and by continuous bubbling with a mixture of 95% O2 and 5% CO2 [10,11]. Each corporal strip was stretched to an optimal isometric tension of 1.0 g and was equilibrated for 90 minutes [12,13]. During the equilibration period, the tissues were washed with fresh Krebs solution every 30 minutes, and the tension was adjusted if necessary. To evaluate the roles of endothelium and Ca2+ channels in RCE-induced relaxation, the endothelial lining of the CC was J Sex Med 2013;10:1720–1729

1722 removed by rubbing the strip between the thumb and index finger for 20 seconds and soaking in 10% 3-[(3-cholamidopropyl)dimethylammonio]1-propane sulfonate (CHAPS) solution for 10 seconds. Successful removal of the endothelium was confirmed in all tissues used by adding acetylcholine (10-6 M) to strips precontracted with phenylephrine (PE) (10-5 M). If the tissue did not respond to acetylcholine, it was accepted as endothelium-free and was enrolled in the study.

Organ-Bath Studies In Vitro In the first series of experiments, to compare the effect of RCE-induced relaxation in CC with udenafil-induced relaxation, the following experiment was conducted. A 10-5 M PE was added to each organ bath containing endothelial intact corporal strips at optimal isometric resting tension after they were equilibrated for 90 minutes. Subsequently, PE resulted in corporal strip contraction, which rapidly reached a steady state of active tension. When a steady state of contraction was achieved, the vasodilatory effect of RCE was studied by cumulative addition of RCE at concentrations ranging from 1.0 to 3.0 mg/mL at the plateau of the PE-induced contraction. Moreover, the relaxation effect of udenafil in concentrations ranging from 10-6 to 10-4 M on PE-induced precontracted cavernosal strips was studied. We compared the magnitude of relaxation between RCE and udenafil. In a second series of experiments, the role of the endothelium in RCE-induced relaxation at concentrations ranging from 1.0 to 4.0 mg/mL was examined by comparing the magnitude of relaxation of PE-induced tone between endotheliumintact and endothelium-denuded specimens. In a third series of experiments, to ascertain whether RCE mediated relaxation through a K+ conductance pathway, we examined the effect of RCE on PE-induced tone after preincubation of strips in 1 mM tetraethylammonium (TEA), a K+ channel inhibitor, for 20 minutes. We compared the magnitude of relaxation between before and after incubation in TEA. In a fourth series of experiments, to delineate the role of inhibition of extracellular Ca2+ influx in RCE-elicited relaxation, cavernosal strips were equilibrated in Ca2+-free Krebs solution containing 0.2 mM Na2-ethylene glycol tetraacetic acid (EGTA) for 30 minutes and washed three times at 10-minute intervals. PE (10-6 M) was added, and then CaCl2 (0.1–2.0 mM) was added at 5-minute intervals to produce a cumulative concentration– J Sex Med 2013;10:1720–1729

Lee et al. response curve of its constrictor effect. When maximum vasoconstriction was achieved, the strip was washed and equilibrated for 30 minutes and then incubated for 10 minutes in RCE of 4 mg/ mL. Concentration–response curves to cumulative addition of CaCl2 were then repeated and compared with their control curves obtained in the same CC. To determine the role of voltage-dependent Ca2+ channels (VDCCs) in RCE-induced relaxation, a fifth series of experiments was conducted. After equilibration, the endothelium-free strips were precontracted with KCl (60 mM), a known VDCC opener [14,15], and once the response had reached the plateau, concentration–response curves to RCE and nifedipine, an L-type Ca2+ channel blocker, were constructed by cumulative addition of the drugs [16]. The relaxations were measured by comparing the developed tension before and after the addition of each substance. The last series of experiments, to clarify whether the relaxation induced by RCE was related to inhibition intracellular Ca2+ release, were carried out in Ca2+-free Krebs solution containing 50 mM EGTA [17,18]. The endotheliumfree strips were washed with Ca2+-free solution three times. After 20-minute incubation with or without RCE 4 mg, PE (10-6 M) was added to stimulate the release of intracellular Ca2+, and the contraction was recorded. The values of the relaxation responses were given as a percentage ratio of the level of tension at the maximal relaxation to the level of maximum contraction by PE.

Cell Cultures Human tissue was obtained from the CC of patients with organic ED undergoing implantation of penile prostheses. Homogeneous explant cell cultures of human corporal smooth muscle (CSM) cells were prepared as previously described [19–22]. The rat vascular smooth muscle cell (VSMC) line A7r5 was purchased from ATCC (Manassas, VA, USA). Cells were maintained in Dulbecco’s modified Eagle’s medium (Gibco Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum and antibiotics (100 units/mL penicillin, streptomycin; Gibco) under 5% CO2 and were passaged every 3–4 days by detaching from the culture dishes with 0.025% trypsin/EDTA (Gibco). Primary human CSM cells were used between passages 2 and 4.

Effect of R. Coreanus Extracts in Corpus Cavernosum

Measurement of [Ca2+]i [Ca2+]i in CSM and A7r5 cells was assessed by measuring Fura-2 fluorescence using dual excitation wavelengths as described previously [23,24]. Briefly, the cells were plated at 1 ¥ 105/mL in 35-mm glass-bottomed dishes and loaded with 5 mM Fura-2 acetoxymethyl ester (AM) and 0.02% pluronic F-127 (Molecular Probes, Invitrogen) at room temperature (22–25°C) in normal physiological salt solution (130 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1.2 mM MgCl2, 5 mM glucose, 10 mM 4-(2-hydroxyethyl)-1piperazineethanesulfonic acid [HEPES], and pH 7.3). After 45-minute incubation, the cells were washed twice and left for 30 minutes at room temperature. Single-cell fluorescence intensities were measured using an Olympus Optical IX70 inverted microscope (Tokyo, Japan) attached to a frame-transfer and back-illuminated chargecoupled device camera (Quantix; Photometrics, Tucson, AZ, USA) using MetaFluor software (Molecular Devices, Sunnyvale, CA, USA). Intracellular [Ca2+] was calculated using the Fura-2 fluorescence ratio (F340/F380) and calibrated using maximum (Rmax) and minimum ratio values (Rmin) obtained by exposing cells to 1 mM ionomycin and 5 mM EGTA (with 0 mM Ca2+ or 10 mM Ca2+), according to the equation described previously [25]. Statistical Analysis The data are expressed as means ⫾ standard error of the mean (SEM), and the value of n refers to the number of separate corporal tissue strips or CSM cells. For statistical analysis, one-way analysis of variance was used, followed by Bonferroni post hoc test or a paired t-test. The Shapiro–Wilk test was used to test for normal distribution of data. P-values less than 0.05 were considered significant. Main Outcome Measures

The main outcome measures are the effects of unripe RCE on ion channels and the intracellular Ca2+ concentration ([Ca2+]i) of CC. Result

Relaxation Effect of Udenafil and RCE on PE-Induced Tone When the contractile response to PE 10-5 M reached a steady level, udenafil or RCE was added cumulatively. Both of them provoked

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Figure 1 Dose–response curves to udenafil (1–100 mM; n = 6; filled squares) and Rubus coreanus (1–3 mg; n = 6; open squares) in rabbit corpus cavernosum strips contracted by phenylephrine (10 mM). Experimental values were calculated relative to the maximal changes from the contraction produced by phenylephrine in each tissue, which were taken as 100%. Data represent the mean ⫾ SE. RCE 1 mg vs. udenafil 1 mM: P > 0.05; RCE 2 mg vs. udenafil 10 mM: P > 0.05; RCE 3 mg vs. udenafil 100 mm: P > 0.05. RCE = Rubus coreanus extract; SE = standard error

concentration-dependent relaxation. The relaxation effect was 17.3 ⫾ 4.3% with udenafil 10-6 M vs. 9.0 ⫾ 5.2% with RCE 1 mg, 37.2 ⫾ 5.0% with udenafil 10-5 M vs. 30.2 ⫾ 5.1 with RCE 2 mg, and 77.9 ⫾ 4.1% with udenafil 10-4 M vs. 70.2 ⫾ 9.0 with RCE 3 mg (n = 6, P > 0.05) (Figure 1).

Role of the Endothelium in RCE-Induced Relaxation Removal of the endothelium from CC strips significantly affected their vasodilator potencies in the presence of 3 and 4 mg of RCEs but did not completely abolish the vasodilator response to RCE. The vasodilator response was 13.2 ⫾ 2.6% at 1 mg, 27.3 ⫾ 3.3% at 2 mg, 41.9 ⫾ 5.0% at 3 mg, and 59.1 ⫾ 7.0% at 4 mg of RCE (n = 6, P < 0.05 at 3.0 and 4.0 mg) (Figure 2). Role of K+ Channels in RCE-Induced Relaxation Tetraethylammonium (TEA) (BKCa and voltagesensitive K+ inhibitor) (1 mM) did not inhibit RCE-induced relaxation in strips precontracted by PE (10-5 M). After pretreatment with TEA, the relaxation effect of RCE was 6.0 ⫾ 3.1% at 1.0 mg/mL, 41.9 ⫾ 8.0% at 2.0 mg/mL, 85.0 ⫾ 12.9% at 3.0 mg/mL, and 111.0 ⫾ 5.4% at 4.0 mg/mL (n = 6, P > 0.05; not shown in the figure). J Sex Med 2013;10:1720–1729

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Effect of RCE on the Sarcoplasmic Reticulum Ca2+ Release Induced by PE In the Ca2+-free solution, PE (10–6 M) induced a transient contraction due to the release of intracellular Ca2+. Pretreatment with RCE (4 mg) for 20 minutes significantly attenuated PE-induced contraction (RCE, 0.43 ⫾ 0.12 g vs. control, 0.06 ⫾ 0.02 g, n = 6, P < 0.05), suggesting that RCE reduced the Ca2+ release from the sarcoplasmic reticulum (Figure 5). Effects of RCE on Basal [Ca2+]i in CSM Cells To identify whether the inhibitory effect of RCE on muscular contraction is related to the attenuaFigure 2 RCE-induced endothelium-dependent or endothelium-independent relaxation in isolated corporal strips. The concentration–response curve of endotheliumintact (E[+], n = 6) or endothelium-denuded (E[-], n = 6) corpus cavernosum strip relaxation following RCE treatment, expressed as a percentage of the PE-induced contraction. Values are expressed as means ⫾ SE. *P < 0.05: E(+) vs. E(-). RCE = Rubus coreanus extract; SE = standard error

Role of Ca2+ Channels in RCE-Induced Relaxation These experiments were performed in Ca2+-free preparations. Priming with PE (10-6 M/L) produced increases of 0.4 ⫾ 0.1 g resting tone, and the subsequent addition of CaCl2 (0.1–2.0 mM) caused stepwise increases in the corporal tone: 21.7 ⫾ 3.2% at 0.1 mM, 46.1 ⫾ 3.6% at 0.3 mM, 78.8 ⫾ 1.9% at 1.0 mM, 91.8 ⫾ 1.4% at 1.5 mM, and 100 ⫾ 0.0% at 2.0 mM. This effect was significantly attenuated by the addition of 4 mg RCE to 4.7 ⫾ 1.7% at 0.1 mM CaCl2, 12.9 ⫾ 4.8% at 0.3 mM CaCl2, 24.7 ⫾ 8.9% at 1.0 mM CaCl2, 36.1 ⫾ 12.0% at 1.5 mM CaCl2, and 43.7 ⫾ 14.2% at 2.0 mM CaCl2 (Figure 3) (n = 6, P < 0.05). To confirm that the inhibition of Ca2+ entry occurred via VDCCs, we performed the following experiments (Figure 4). When the contractile response to high K+ (60 mM) reached a steady level, nifedipine (a well-known L-type Ca2+ channel blocker) or RCE was added cumulatively. Both of them provoked concentration-dependent relaxation. The maximal relaxation was 122.8 ⫾ 14.2% with nifedipine 10-5 M vs. 91.8 ⫾ 9.4% with RCE 4 mg (Figure 4) (n = 6). The observation that KCl-induced contraction was the result of Ca2+ influx through VDCCs suggests that RCE prevents Ca2+ influx via VDCCs in CSM. J Sex Med 2013;10:1720–1729

Figure 3 Effects of RCE at 4 mg on the Ca2+-induced (0.1– 2 mM) contraction of rabbit corpus cavernosum strips pretreated with phenylephrine (1 mM). Data are expressed as means ⫾ SE (n = 6). *P < 0.01: 4 mg RCE vs. control. RCE = Rubus coreanus extract; SE = standard error

Figure 4 Concentration–response curves for RCE-induced relaxation in high-K+ (60 mM) precontracted rabbit corpus cavernosum strips (without endothelium) in response to nifedipine (open squares, n = 6) or RCE (closed squares, n = 6). RCE = Rubus coreanus extract

Effect of R. Coreanus Extracts in Corpus Cavernosum

Figure 5 Inhibitory effect of RCE on the PE (1 mM)-induced contraction in Ca2+-free solution. Results are presented as mean ⫾ SE. n = 6. *P < 0.01 compared with control. RCE = Rubus coreanus extract; PE = phenylephrine; SE = standard error

tion of [Ca2+]i, we examined its effects on [Ca2+]i using Fura-2 ratiometric Ca2+ imaging. As shown in Figure 6, application of 0.1% DMSO to the cells as a vehicle control had no effect on [Ca2+]i (control: 136.8 ⫾ 8.8 nM, 0.1% DMSO: 135.4 ⫾ 10 nM, n = 9) in the presence of normal extracellular Ca2+. By contrast, application of RCE (1, 10, or 100 mg) significantly reduced the basal [Ca2+]i in a dose-dependent manner (1 mg: 123.4 ⫾ 10.0 nM, P > 0.05; 10 mg: 117.9 ⫾ 5.0 nM, P < 0.05; and 100 mg: 81.7 ⫾ 8.0 nM, n = 9, P < 0.05 vs. 0.1% DMSO). At higher concentration, RCE (100 mg) had a greater inhibitory effect, which was rapidly and fully reversible after washout.

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Inhibition of KCl-Induced [Ca2+]i Increases by RCE in A7r5 Cells To assess the effects of RCE on Ca2+ signaling in more detail, we employed a commonly used rat VSMC line, A7r5 cells. We examined whether Ca2+ influx through VDCCs was affected by RCE. Bath application of 60 mM KCl evoked a remarkable and reproducible [Ca2+]i elevation from a basal level of 120.3 ⫾ 5.5 to 580.8 ⫾ 142.7 nM (n = 5), a 382.8% increase. In the same cells, pretreatment with RCE for 5 minutes caused a dose-dependent reduction of basal [Ca2+]i and a subsequent highK+-induced [Ca2+]i response, reaching statistical significance at the 100 mg/mL of RCE concentration (Figure 7A). The high-K+-induced peak [Ca2+]i was inhibited by approximately 26.1% at 10 mg/mL of RCE (P < 0.05) and 59.8% at 100 mg/mL of RCE (P < 0.05). After washout with normal physiologic salt solution, the second application of KCl also increased [Ca2+]i to a similar extent as the first application. These data are summarized in Figure 7B and indicate that RCE modulates [Ca2+]i in part by inhibition of Ca2+ influx through VDCCs. Inhibition of AVP-Induced [Ca2+]i Increases by RCE in A7r5 Cells To further investigate the effect of RCE on smooth muscle cell Ca2+ signaling, we determined the effects of RCE on the response to [Arg8]vasopressin (AVP), which induces Ca2+ release from the sarcoplasmic reticulum in VSMCs [26]. Application of 20 nM of AVP evoked a statistically

Figure 6 Effects of RCE on the basal [Ca2+]i of hCSM cells. RCE (1–100 mg/mL) was added to the cells in the presence of extracellular Ca2+ (2 mM). The [Ca2+]i of a single cell was dose-dependently decreased. (A) Representative tracing of the [Ca2+]i response evoked by various concentrations of RCE (1–100 mg/mL). The right panel shows summarized data of the RCE-induced changes in [Ca2+]i in hCSM cells. (B) Summary of the means ⫾ SE of basal [Ca2+]i in RCE-treated CSM cells. n = 9. *P < 0.05 vs. 0.1% DMSO control. DMSO = dimethyl sulfoxide; hCSM = human corporal smooth muscle; RCE = Rubus coreanus extract; SE = standard error

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Figure 7 Inhibitory effects of RCE on the 60 mM K-induced [Ca2+]i increase in A7r5 cells. Cells were pretreated with 10 or 100 mg/mL of RCE for 5 minutes before 60 mM KCl application. (A) A representative tracing of the [Ca2+]i response evoked by 60 mM KCl alone or 60 mM KCl plus RCE. RCE produced a concentration-dependent reduction of the 60 mM KCl responses. (B) Left panel shows a summary of the means ⫾ SE of basal [Ca2+]i in RCE-treated A7r5 cells. Right panel shows a summary of the effects of RCE-induced inhibition of the 60 mM KCl induction of [Ca2+]i. n = 5. *P < 0.05 vs. control. RCE = Rubus coreanus extract; SE = standard error

significant [Ca2+]i elevation (from a basal level of 138.9 ⫾ 8.5 to 620.5 ⫾ 154.8 nM, n = 14). The extent of this increase was similar to that induced by KCl. However, pretreatment with 100 mg of RCE for 10 minutes significantly inhibited the AVP-induced [Ca2+]i increase (Figure 8A). At a concentration of 100 mg/mL of RCE, the inhibitory effect was approximately 64.0% (P < 0.05). These data are summarized in the bar graph in Figure 8B and suggest that RCE may cause relaxation of CSM in part by altering intracellular J Sex Med 2013;10:1720–1729

Ca2+ by decreasing Ca2+ release from the sarcoplasmic reticulum. Discussion

In the present study, RCE effectively relaxed PE-induced tone in rabbit CC, and the relaxation effect was dose dependent and similar to that of udenafil (Figure 1). Our data show that 3 mg/mL of RCE was comparable with 10-4 M of udenafil. A similar result has been reported in a recent study

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Figure 8 Inhibitory effects of RCE on the AVP-induced [Ca2+]i increase in A7r5 cells. (A) Representative tracing of the [Ca2+]i response evoked by AVP alone (left panel) or AVP plus 100 mg/mL of RCE (right panel). (B) Bar graph representing the average peak change in [Ca2+]i induced by AVP in the absence and presence of RCE. Data are expressed as the mean ⫾ SE. *P < 0.05 relative to the respective control (paired Student’s t-test). n = 14. AVP = [Arg8]-vasopressin; RCE = Rubus coreanus extract; SE = standard error

using the rabbit CC [8]. Our study confirms those results as well as the potency of RCE. That study [8] indicated that RCE increased cGMP and cyclic adenosine monophosphate (cAMP) concentrations and increased the expression of endothelial nitric oxide synthase (eNOS) and neuronal nitric oxide synthase (nNOS) in the rabbit CC. To ascertain whether activation of the NO-cGMP pathway was involved in the relaxation signal pathway, those authors investigated RCEevoked relaxation after treatment with ODQ (selective inhibitor of guanylate cyclase) or L-NAME (nonselective inhibitor of NO). However, neither agent completely inhibited the RCE-induced relaxation. Thus, it was speculated that RCE-induced relaxation involves other pathways independent of the endothelium. In the present study, removal of the endothelium did not completely abolish the vasodilator response of RCE (Figure 2), consistent with the previous study.

Hence, our study focused on another pathway to modulate cavernosal tone, involving direct action on smooth muscle cells. Such a mechanism could involve opening potassium channels or inhibiting the opening of Ca2+ channels. In this study, TEA (BKCa and voltage-sensitive K+ channel inhibitor) (1 mM) did not inhibit RCEinduced relaxation in strips precontracted by PE (10-5 M) in organ-bath experiments. In addition, our preliminary study using the patch clamp technique showed that RCE did not activate BKCa channels (data not shown). These results suggest that the relaxation effect of RCE is not related to potassium channels. In contrast to this, the CaCl2induced constriction of corpus cavernosal strips bathed in Ca2+-free buffer and primed with PE was abolished by RCE. In addition, RCE decreased basal [Ca2+]i in CSM cells. These results provide evidence that the relaxation effect of RCE is related to the inhibition of intracellular Ca2+ increase. J Sex Med 2013;10:1720–1729

1728 The rat VSMC line A7r5 is usually used for intracellular calcium homeostasis as a substitute smooth muscle cell for primary smooth muscle cells, such as human CSM cells [27,28]. We confirmed that increases of [Ca2+]i evoked by 60 mM K+-containing solution in A7r5 cells were suppressed by RCE in a concentration-dependent manner. RCE relaxed KCl-induced tone in endothelium-free CC. Both of these results indicate that RCE blocks VDCCs. A7r5 cells express two distinct types of VDCC, the L type and the T type [29], and among all of the VDCCs, only L-type voltage-gated Ca2+ channels exist in the smooth muscle of the CC [30,31]. The relaxation effect of RCE on KCl-induced tone in endothelium-free CC was comparable to that of nifedipine (an L-type Ca2+ channel blocker). Taken together, these results suggest that RCE blocks Ca2+ influx via L-type voltage-gated Ca2+ channels. We also investigated another pathway by which intracellular Ca2+ is increased: the release of Ca2+ from sarcoplasmic reticulum. RCE decreased basal [Ca2+]i and the AVP-induced [Ca2+]i increases in A7r5 cells. AVP, a pituitary hormone, induces Ca2+ release from the sarcoplasmic reticulum and Ca2+ influx via multiple Ca2+-permeable nonselective cation channels (transient receptor potential cation channels [TRPC6] channels) in VSMCs [32]. In our patch clamp study, we confirmed that RCE had no effect on carbachol-induced TRPC6 channel activity in TRPC6 overexpressing human embryonic kidney (HEK) cells (data not shown). In addition, RCE inhibited the contraction of endothelium-free CC induced by PE in Ca2+-free solution. PE causes cavernosal contraction by stimulating the release of Ca2+ from sarcoplasmic reticulum. Contraction of cavernosal strips by PE in Ca2+-free solution is solely due to release of Ca2+ from sarcoplasmic reticulum [17,18]. Hence, RCE might act as a modulator of CSM tone by inhibiting Ca2+ release from sarcoplasmic reticulum. New experiments are now focusing on a telemetry model to evaluate the efficacy of RCE. We found that there is a tendency to recovery of ED in diabetic rats. The following experiments are ongoing to ensure statistical significance. Additionally, we plan to collect quantitative data related to eNOS and nNOS, playing important roles in the physiology of erectile function. Conclusions

In conclusion, we found that RCE relaxes the CC via an endothelium-independent pathway and may J Sex Med 2013;10:1720–1729

Lee et al. inhibit VDCCs and Ca2+ release from sarcoplasmic reticulum. Our findings should be valuable in the development of new drugs for ED using natural products. Acknowledgments

This study was supported by a grant from the Korean Health Technology R&D Project, Ministry for Health, Welfare & Family Affairs, Republic of Korea (A111535). The author(s) report no conflicts of interest. Corresponding Author: Sung Won Lee, MD, PhD, Department of Urology, Samsung Medical Center, Sungkyunkwan University School of Medicine, #50 Ilwon-dong, Kangnam-ku, Seoul 135-710, Korea. Tel: (2) 3410-3552; Fax: (2) 3410-3027; E-mail: drswlee@ skku.edu References 1 Feldman HA, Goldstein I, Hatzichristou DG, Krane RJ, McKinlay JB. Impotence and its medical and psychosocial correlates: Results of the Massachusetts Male Aging Study. J Urol 1994;151:54–61. 2 Araña Rosaínz Mde J, Ojeda MO, Acosta JR, Elías-Calles LC, González NO, Herrera OT, García Álvarez CT, Rodríguez EM, Báez ME, Seijas EÁ, Valdés RF. Imbalanced low-grade inflammation and endothelial activation in patients with type 2 diabetes mellitus and erectile dysfunction. J Sex Med 2011;8:2017–30. 3 NIH Consensus Conference. Impotence. NIH consensus development panel on impotence. JAMA 1993;270:83–90. 4 Porst H. The future of erectile dysfunction (ED). Arch Esp Urol 2010;63:740–7. 5 Supuran CT, Mastrolorenzo A, Barbaro G, Scozzafava A. Phosphodiesterase 5 inhibitors—Drug design and differentiation based on selectivity, pharmacokinetic and efficacy profiles. Curr Pharm Des 2006;12:3459–65. 6 Jiann BP, Yu CC, Su CC, Tsai JY. Compliance of sildenafil treatment for erectile dysfunction and factors affecting it. Int J Impot Res 2006;18:146–9. 7 Lee SJ. Korean fork medicine. Seoul: Seoul National University Press; 1966. 8 Zhao C, Kim HK, Kim SZ, Chae HJ, Cui WS, Lee SW, Jeon JH, Park JK. What is the role of unripe Rubus coreanus extract on penile erection? Phytother Res 2011;25:1046–53. 9 Sohn DW, Kim HY, Kim SD, Lee EJ, Kim HS, Kim JK, Hwang SY, Cho YH, Kim SW. Elevation of intracavernous pressure and NO-cGMP activity by a new herbal formula in penile tissues of spontaneous hypertensive male rats. J Ethnopharmacol 2008;120:176–80. 10 Kam SC, Do JM, Choi JH, Jeon BT, Roh GS, Hyun JS. In vivo and in vitro animal investigation of the effect of a mixture of herbal extracts from Tribulus terrestris and Cornus officinalis on penile erection. J Sex Med 2012;9:2544–51. 11 Capel RO, Mónica FZ, Porto M, Barillas S, Muscará MN, Teixeira SA, Arruda AM, Pissinatti L, Pissinatti A, Schenka AA, Antunes E, Nahoum C, Cogo JC, de Oliveira MA, De Nucci G. Role of a novel tetrodotoxin-resistant sodium channel in the nitrergic relaxation of corpus cavernosum from the South American rattlesnake Crotalus durissus terrificus. J Sex Med 2011;8:1616–25.

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