Eight while male rabbits (New Zealand, 2.5~3.0 kg) were used. The penis was resected and sections of the CC (2×2×10 mm) were prepared. The thoracic aorta was resected by removing the connective tissue, and the ring (3 mm) was prepared. The CC sections were pretreated with 3 mL of 0.3% 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS) to detach vascular endothelial cells, manually rubbed for 20 seconds, and then rinsed with Krebs-Henseleit (KH) solution. Vascular endothelial cells were removed by smoothly rubbing the inside of the vessel of the aortic ring with a 25-gauge needle tip. The removal of vascular endothelial cells was confirmed by checking that CC section contraction induced by phenylephrine (PE; 5×10⁻⁶ mol) was not prevented by acetylcholine (10⁻⁵ mol).

The CC sections and the aortic rings were transferred to the organ bath. One end was connected to the muscle fixation ring and the other end was connected to the isometric tension transducer, and signals were observed and measured via physiography. Chart 5 software (ADInstruments, Bella Vista, Australia) was used to monitor the tension in real time. The KH solution in the organ bath was maintained at 37℃ and a pH of 7.4 by continuously supplying air consisting of 95% O₂ and 5% CO₂. The initial tension in each section was maintained at approximately 2 g, and the KH solution was replaced every 30 minutes for 2 hours to reach the resting state. The degree of contraction was observed by adding PE after reaching the resting state. Each section was gradually reacted with the PE and increased to the optimal point of the length-tension curve, as determined by the active tension change. The resting state was restored by rinsing with the KH solution 3 times. The tissues were contracted for pretreatment with PE in the organ bath while maintaining the resting optimal isometric tension.

The relaxation response was investigated to assess the CC relaxation mechanism induced by alcohol (0.01%~2%) and acetaldehyde (0.001%~0.1%). The CC was left in the KH solution with the pretreatment drug. The tissues were then contracted with PE to reach the plateau. The following drugs were used for pretreatment: N(G)-nitro-L-arginine methyl ester (L-NAME) (10⁻⁵ mol), propranolol (10⁻⁵ mol), indomethacin (3×10⁻⁵ mol), glibenclamide (10⁻⁵ mol), 4-aminopyridine (10⁻⁵ mol), and methylene blue (10⁻⁵ mol).

Forty male white rats (Sprague-Dawley, 280~320 g) were classified into 5 groups depending on the amount of hours that had passed after alcohol administration (n=8 in each group). The rats were placed in the supine position, and blood pressure (BP) was monitored with a polyethylene-50 tube in the carotid artery. The prostate was exposed via a midline incision. In addition, the pelvic nerve plexus was checked. The penis was dissected to the penile crura. The ischiocavernosal muscle around the penile crura was dissected to assess the tunica albuginea in the penis, and a 25-gauge polyethylene-50 tube was inserted in the CC.

The mean arterial pressure (MAP) and intracavernosal pressure (ICP) were measured by the BP manometer transducer and recorder. An electric stimulus was provided with 5 volts for 60 seconds. The pressure before, during, and after the stimulus were measured to assess the hemodynamic response to nerve stimulation. The maximum ICP/MAP percentage was measured by stimulating the cavernosal nerve to assess the response induced by the nerve stimulation and compared across groups.

The penile tissues in the control group and the 2-hour alcohol group were quickly frozen in liquid nitrogen and homogenized in ice-cold 6% trichloroacetic acid. The homogenized tissues were centrifuged for 15 minutes and the cellular proteins in the supernatant were separated in the usual manner. The samples were classified into the control group and the 2-hour alcohol group, and the concentrations of secreted cyclic adenosine monophosphate (cAMP) (n=8) and cyclic guanosine monophosphate (cGMP) (n=8) were measured with an immunoassay. The immunoassay was based on the antagonistic binding method, in which horseradish peroxidase-labeled cAMP and cGMP and fixed amounts of the sample cAMP and cGMP competed for the polyclonal antibody position of the rabbit. The polyclonal antibody was combined to a microplate treated with goat anti-rabbit antibody during the response. It was rinsed to remove excessively binding and non-binding components of the sample, and the substrate solution was added to determine the binding enzyme activity. Absorbance was measured at 450 nm.

Twelve male white rats were classified into a control group and a 12-week alcohol group (n=6 in each). As an animal model of chronic alcohol administration, 15% alcohol (3 g/kg) was orally administered every day for 12 weeks in the 12-week alcohol group. The same amount of saline was administered with the same method for 12 weeks in the control group. Twenty-four hours after the last administration at 12 weeks, the ICP/MAP percentage was measured using the same method discussed above for the acute alcohol study.

Changes in the concentration of cAMP and the cGMP in the CC in the control group and the 12-week alcohol group were observed. Twenty-four hours after the last alcohol administration at week 12, levels of cAMP and cGMP were measured using the same method discussed above for the acute alcohol study.

The mid-part of the penile section in each group was excised and fixed with 10% formalin phosphate-buffered solution. It was then embedded in paraffin, and the samples were excised with a thickness of 4 µm and dyed with Masson trichrome (n=6 in each group).

Collagen was measured using the Sircol collagen assay kit. The frozen penile tissue was excised at thicknesses of 2~3 mm, left in small tubes, mixed well with 1 mg of pepsin including 0.5 mol acetic acid for each 10 mg of tissue and left overnight at 4℃. The next day, the tissues were centrifuged for 60 minutes at 15,000 rpm, the supernatant was filtered with an 0.8-µm pore-size filter, and 200 µL was mixed with 1 mL of Sircol dye reagent and shaken well at room temperature for 30 minutes. It was then centrifuged again, the supernatant was removed, and the remaining material was mixed with 1.0 mL of 0.5 N NaOH, mixed for 5 minutes, and the collagen concentration was measured with a microwell plate reader in the 550-nm range.

Sections of CC stored at −70℃ were sequentially precipitated with 10%, 20%, and 30% of sucrose solutions to fix the tissues, and the tissue sections 10 µm in thickness were prepared with an electric frozen slicer. The specific antibody against endothelial nitric oxide synthase (eNOS) was used with a dilution ratio of 1:200 as the primary antibody. The antibody was detected using the avidin-biotin peroxidase technique with diamino benzidine as the chromogen, and the counterstain was performed with H&E.

The tissues in the 2 CC groups were homogenized using 0.1 mol phosphate-buffered saline (potassium phosphate sodium, pH 7.4) at 4℃ and dissolved in a lysis buffer (5 mmol/L of glycerophosphate, 2 mmol/L of MgCl₂, 1 mmol/L of ethylene glycol-bis(β-aminoethyl ether)-N,N, N′,N′-tetraacetic acid (EGTA), 0.5% Triton X-100, 0.5% NP-40, 1 mmol/L of dithiothreitol, and 100 mL of a protease inhibitor cocktail). The protein concentration was measured using a protein assay kit (Bio-Rad, Hercules, CA, USA). Each lane of a sodium dodecyl sulfate polyacrylamide gel was loaded with 25 µg of protein to perform electrophoresis. The separated protein was transferred to a nitrocellulose membrane with a semi-dry transfer unit, and was treated with a blocking solution (5% skim milk/tris-buffered saline-tween [TBST]) at 4℃ overnight to block the intrinsic factors. The eNOS antibody was used as the primary antibody, was allowed to sit for 2 hours, and then the samples were rinsed 3 times for 15 minutes with TBST and treated with the secondary antibody (horseradish peroxidase conjugated goat anti-rabbit immunoglobulin G) for 1 hour. The samples were then developed by being exposed to hyperfilm after a color reaction with a chemiluminescent reagent. The protein standard molecular marker conformed to precision protein standards, the density of each protein was measured using an image analysis program, β-actin density was measured for calibration, and the extent of expression of each protein was surveyed.

Statistical significance in the acute alcohol group was assessed using analysis of variance, and comparisons were made with the chronic alcohol group using the Student's t-test. All statistical analyses were performed in PASW Statistics ver. 18.0 (IBM Co., Armonk, NY, USA). p<0.05 were considered to indicate statistical significance.