1 Introduction

Hyperlipidemia has been considered one of the main risk factors of cardiovascular diseases and vasculogenic erectile dysfunction as well. Among the lipid fractions, hypercholesterolemia has been known to be the greatest risk. The level of blood cholesterol is now on the rise in Asia because of the increasing adoption of a western diet, thus, hypercholesterolemia has recently aroused a lot of public attention in this area (Table 1).

Table 1. Changes of mean level of total cholesterol in blood of normal Korean adults.

2 Incidence of hyperlipidemia in impotent patients^[5]

The incidence of hyperlipidemia was investigated in men aged over 40 years with erectile dysfunction. Blood levels of the various lipid fractions were measured in 943 patients with erectile dysfunction and 242 normal men (Table 2).  

Table 2. Incidence of abnormally high or low blood level of lipid fractions in impotent patients and normal men, 40-70 years old. ^bP<0.05, ^cP<0.01 vs control.

3 Erection response to intracavernous injection of papaverine in impotent patients with hyperlipidemia^[7]

4 Relaxation responses of isolated cavernosal smooth muscles to endothelium-dependent and endothelium-independent vasodilators in impotent patients with hypercholesterolemia or hypertriglyceridemia^[8]

To investigate the relaxation response of the cavernous smooth muscle to endothelium-dependent and endothelium-independent vasoactive agents, cavernous strips taken from the impotent patients with hypercholesterolemia (n=5) or hypertriglyceridemia (n=6) during penile prosthetic surgeries were placed in organ chamber and isometric tension study was conducted. There was no difference in contractile response induced by various concentrations of  norepinephrine (10^-9 to 10^-4 mol/L) between the impotent patients with or without hypercholesterolemia or hypertriglyceridemia.

The relaxation responses of the tissues taken from the patients with hypercholesterolemia to endothelium-dependnent vasoactive agents, acetylcholine (10^-9-10^-4 mol/L), adenosine (10^-9-10^-4 mol/L), and high dose bradykinin (10^-6-10^-5 mol/L) were significantly reduced, as compared to those from the patients with normal blood level of the total cholesterol (n=16). On the other hand, there was no significant difference in the relaxation responses of the tissues to the endothelium-independent vasodilator, papaverine (10^-6-10^-5 mol/L) and verapamil (10^-6-10^-5 mol/L) between the two groups.

5 Involvement of superoxide radical in the impaired endothelium-dependent relaxation of cavernous smooth muscle in hypercholesterolemic rabbit^[9]

It was reported that endothelial and smooth muscle cells, neutrophils and monocytes or platelets might be a source of oxygen free radical, which were known to interact with polyunsaturated fatty acids of biological membrane, thus leading to lipid peroxidation^[10]. Oxygen free radicals such as superoxide radical (O₂^- ), hydrogen peroxide (H₂O₂), hydroxyl radical (OH) and singlet oxygen (¹O₂) are generated by a partial reduction of oxygen during the biological processes^[11]. Among the free radical species, superoxide radical had the main role of causing oxygen toxicities. The first line of defense against free radicals is SOD, which scavenges the superoxide radical. The next line of defense are the glutathione peroxidases and catalases. It was reported that SOD activities were generally increased in accordance with the activity of superoxide radical, but little information was reported about the changes in catalase and glutathione peroxidase activity.

Henriksson et al^[12] found evidence of a generation of free radicals in hyperlipidemia and atherosclerosis, and relationships between  the free radicals and scavengers, SOD  and catalase. The SOD increased in atherosclerotic aorta by feeding a high cholesterol diet. Gryglewski et al^[13] reported that the superoxide radicals contributed significantly to the destruction of the EDRF, and that the EDRF was protected from breakdown by SOD and Ca²⁺, but not by catalase. Del Boccio et al^[14] reported that the changes in aortic antioxidant defense mechanisms and lipid peroxidation precede the massive vascular lipid infiltration in cholesterol-fed rabbits. And some antioxidant mechanisms were increased (SOD, glutathione peroxidase and total thiol compounds), whereas others were depressed (catalase, glutathione reductase and glutathione transferase), thus potentially reducing or increasing vascular susceptibility to oxidative injury.

In our study, the production of superoxide radicals and the activities of total SOD, Mn-SOD and Cu, Zn-SOD increased significantly in the hypercholesterolemic group compared with the control group. The activities of catalase and glutathione peroxidase also increased in the hypercholesterolemic group, but were not significantly higher than in the control group. This suggests that production of the superoxide radicals in cavernous tissues increases in the state of hypercholesterolemia, which leads to functional impairment of cavernous smooth muscle relaxation in response to endotheliummediated stimuli. SOD seems to play a major role as a scavenger against the superoxide radical in cavernous tissue.

6 Effects of isolated lipoproteins and TG, combined oxidized LDL plus TG, and combined oxidized LDL plus HDL on contractile and relaxation response of rabbit  cavernous smooth muscles^[15]

LDL is oxidized by oxygen free radicals from the arterial endothelium and the smooth muscle cells, and the oxidized LDL is toxic to endothelia. LDL peroxidation participates in early stages of the atherosclerosis, and injuries of the endothelial cells have principal role in progression of the atherosclerosis. Recent studies showed that oxidized LDL inhibited endothelium-dependent relaxation and enhanced contraction of the vascular smooth muscle^{[16, 17]}. Although controversial, native LDL also might cause alterations in vasoactivity. 

The effects of TG, not to speak of the synergistic effects of combined TG and LDL on contractile and relaxation response of vascular and cavernous smooth muscles have not been established to date. Impaired relaxation response to acetylcholine was demonstrated in hypertriglyceridemic humans with normal levels of plasma LDL-cholesterol, although contrary findings were also reported^[3,4].

We investigated the effects of isolated lipoproteins and TG, and the effects of combined oxidized lipoproteins plus TG and the combined LDL plus HDL on the contractility and relaxation responses of rabbit cavernous smooth muscle. Cavernous muscle strips taken from 40 male New Zealand White rabbits (2.5-3.0 kg; n=40) were studied in organ chambers for isometric tension measurement. The strips were exposed to HDL (50 g/mL) , LDL (50 g/mL), oxidized LDL (50 g/mL), TG (short-, medium-, and long-chain; 2.5 mg/mL), and combined oxidized LDL plus TG and combined oxidized LDL plus HDL for 30 min. The oxidized LDL was obtained by exposure of LDL to copper ion for 24 hours at room temperature.

Both HDL and LDL did not affect contraction and relaxation responses of the cavernous muscles. The oxidized LDL did not affect norepinephrine (10^-9-10^-4 mol/L) induced contractility of the strips, but significantly (P<0.05) decreased the relaxation response of the cavernous strips precontracted with norepinephrine (10^-4 mol/L) to endothelium-dependent agonist, acetylcholine (ACh, 10^-9- 10^-4 mol/L) (Figure 1). The relaxation responses to endothelium-independent vasodilator, sodium nitroprusside (SNP) were not changed after treatment with the oxidized LDL (Figure 1). Nonspecific NO synthase inhibitor (L-NAME) completely inhibited the relaxation response to ACh, and L-arginine partially improved the diminished relaxation, which suggests that the oxidized LDL may inhibit endothelium-dependent, NO-mediated relaxation.

Figure 1. Effect of oxidized LDL on relaxation response of rabbit cavernous smooth muscle. Acetylcholine-induced relaxation was significantly (P<0.05) impaired in oxidized LDL-treated muscles, whereas sodium nitroprusside-induced relaxation was not.

TG significantly (P<0.05) decreased the contractile response of cavernous muscle to NE, regardless of the type of TG, compared to the control group. Short-chain TG attenuated norepinephrine-induced contraction by 60%, medium-chain by 45%, and long-chain by 73%. No significant change of the relaxation responses to both endothelium-dependent and endothelium-independent vasodilators was noted in the short, medium or long chain TG-treated strips, although only the relaxation response to high dose SNP was significantly (P<0.05) decreased in the short-chain TG groups.

We could not find any significant synergistic nor detoxication effects of the oxidized LDL or HDL on the contraction and relaxation responses when they were added to TG.

7 Involvement of e-NOS in the impaired endothelium-dependent relaxation of cavernous smooth muscle in hypercholesterolemic rabbit^[18]

It has been suggested that the impairment of endothelium-dependent relaxation in hypercholesterolemia and atherosclerosis might be due to impaired ability to synthesize or release NO, which is an endothelium-derived factor^[19]. Kanazawa et al^[20] reported that the mechanism of impaired endothelium-dependent relaxation in atherosclerotic aorta of hypercholesterolemic rabbits was not due to the down-regulation of eNOS mRNA and protein. However, functional activities of eNOS could be altered even though the expression of eNOS mRNA and protein was maintained. So, we designed a study to evaluate whether functional impairment and/or protein expression of cNOS was involved in the impairment of endothelium-dependent relaxation of cavernous smooth muscle in hypercholesterolemic rabbits.

Sixty male New Zealand White rabbits were randomly divided into control and experimental groups. The control group (n=20) received a regular diet, while the two experimental groups (n=20 for each) were fed a 2% cholesterol diet for 4 and 8 weeks, respectively. We conducted isometric tension studies with endothelium-dependent and endothelium-independent vasodilators with or without preinocculation with L-arginine and NANC-selective electrical field stimulation on isolated strips of corpus cavernosum. Expression of cNOS protein was assessed by Western blot analysis. cNOS activities in both cytosolic and particulate fractions were measured by determining the conversion of L-arginine to L-citrulline.

Blood levels of cholesterol were 28-fold higher (P<0.01) in the experimental groups than in the control group. The relaxation responses to endothelium-dependent agents (acetylcholine, adenosine 5-diphosphate) were significantly reduced (P<0.05) in both experimental groups, regardless of their incubation with L-arginine, compared with the control group. However, no differences were found among the three groups in the relaxation response to endothelium-independent agents (papaverine and nitroprusside) and to NANC-selective electrical field stimulation.

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