M. Shiono

polyclonal antibody revealed the presence ofnNOS in the nerves. The staining in 14-weekold rats was strong (Fig. 2A), and the staining in
15-month-old rats with testosterone
replacement was almost the same in level as in
14-week-old rats (Figs. 2D and E). In 15month-old rats without testosterone
replacement, nNOS was weakly positive (Fig.
2C).

Recently, several physiological studies(Holmquist et al., 1991; Burnett et al., 1992) ofelectrically induced erections as well as pharmacological studies (Ignarro et al., 1990; Kimet at, 1991; Rajifer et al., 1992) have indicatedthat NO plays a major role in the mediation ofpenile erection. Penile erection results fromrelaxation of the corporal smooth muscle andpenile cavernosal arteries. NO mediates thisprocess as supported by evidence showing thatNO has a direct vasodilatory effect on cavernosal tissue and that relaxation of penile smoothmuscle is prevented by NOS inhibitors (Kraneet al., 1989; Seftel et al., 1994; Burnett, 1997).
The production of NO is mediated by a familyof NOS that all represent distinct gene products(Forstermann et al., 1995). There are 3 iso-Discussion
The results of this study using young adult andmiddle-aged rats showed that there was an agerelated decrease in serum testosterone concentration, NO level and NOS expression in thecorpus cavernosum, and moreover, thatin middle-aged rats with 2 months of
testosterone replacement which wascontinued until the rats reached the ageof 15 months, those levels wererestored to the level in young adult rats.
Several reports show that a low
testosterone level decreases penile
erection and treatment with testosterone restores it (Mills et al., 1992;
Heaton and Varrin, 1994; Meisel and A
Sachs, 1994; Garban et al., 1995). One
important question is what is the role of
testosterone by itself in themaintenance of penile erection. The
author has significantly extended theseprevious findings by demonstratingthat the most likely mechanism of
penile dysfunction is an aging-induced
reduction in the level of penile NO andNOS, a reduction which is prevented
by testosterone.

B

A: G1, young adult rat (14-week-old)
B: G2, middle-aged rat (13-month-old)

Fig. 2. Sections of rat corpus cavernosum tissue immunostained by ant-neuronal nitric oxide synthase(nNOS) antibodies (original magnification × 200).
A, D and E: Nerve fibers are stained at the same level in G1, G4 and G5.
C: Staining is weaker than that of the young adult group.
[Figs. 2A and B on p. 50 and Figs. 2C–E on p. 51]

50

Erectile dysfunction with aging in rats

forms, 2 constitutive and 1 inducible, which differ in their dependence on intracellular calcium.
Of the constitutive isoforms, eNOS was first
identified in bovine aortic endothelial cells andappears to play an important role in the transduction of signals from the bloodstream to theunderlying smooth muscle which causes vasorelaxation (Lamas et al., 1992; Sessa et al.,1992). The expression of eNOS protein andmRNA has been examined in several disease
processes. Increased expressions of eNOSmRNA and protein has been found in atherosclerotic vessels and in response to shear stressas well as estrogen (Kanazawa et al., 1992;
Nishida et al., 1992; Weiner et al., 1994). Increased eNOS activity has been found in causesof renal failure, whereas decreased
activity has been found in pulmonary
hypertension (Weiner et al., 1994;Conger et al., 1995). These suggestthat the eNOS expression is varied
and that its expression is a functionof the offending disease process.
eNOS has been thought to be a putative key enzyme of penile erection inthat acetylcholine stimulates the release of endothelium-derived relaxation factors from intact rabbit cavernosal smooth muscle cells (Seftel etal., 1994) and also induces the relaxation of penile smooth muscle, a condition which is reversed by NOS inhibitors (Kim et al., 1991). Furthermore, impaired endothelium-dependent corporal smooth muscle relaxation has been found in diabetic menwith erectile dysfunction (Saenz etal., 1989). The recent immunohistochemical localization of eNOS in theendothelial layers of the dorsal penile
arteries, veins and corporal sinusoids
places the enzyme in a physiologically critical location for mediating
penile erection (Burnett et al., 1996).
nNOS has been localized in the
rat penile autonomic nerves, theadventitia of rat penile arterioles, andthe autonomic innervation of thehuman penis (Burnett et al., 1993). It
has also been reported that there areCDE

C: G3, middle-aged rat
D: G4, low-dose testosterone replaced
middle-aged rat
E: G5, high-dose testosterone replaced
middle-aged rat(G3–G5: 15-month-old)

Figs. 2C–E. Continued from the previous page.

51

M. Shiono

numerous NOS-containing nerve fibers andterminals innervating corpus cavernosumsmooth muscles and vessels in the spaces of thecorpus cavernosum.
The presumed role of nNOS in penile erection has been studied in rats by using electricalstimulation of the pelvic nerves, which caused
penile erection that was prevented by the NOS
inhibitor L-nitroarginine methyl ester (Burnettet al., 1992, 1996; Bush et al., 1992). The NOS
activity (presumed to be nNOS) has been indirectly measured in penile tissue homogenates
by using a [ 3H]- L -arginine to [3H]-citrulline
conversion assay, which, along with Western
blotting analysis, showed a decrease in penile
NOS activity and penile nNOS protein content
in diabetic rats with erectile dysfunction(Vernet et al., 1995). A recent demonstrationthat nNOS-deficient transgenic mice maintain anormal penile erection throughout eNOSdependent production of NO suggests thateNOS may also have an important primary andcompensatory role in mediating penile erection(Burnett et al., 1996).
Previous studies have shown a decreased
penile NOS activity in models of erectile dysfunction, such as hypogonadism, androgen receptor blockade and diabetes (Burnett et al.,1992). Our model of middle-aged rats had alow level of the penile NOS expression. Lugget al. (1995) reported that in rats, aging induced
a considerable reduction in the erectile responseto electric field stimulation which was accompanied by a decrease in NOS activity in very oldrats. Several reports have shown that a low testosterone level decreases penile erection andtestosterone restores it (Mills et al., 1992; Heaton
and Varrin 1994; Meisel and Sachs, 1994; Garban
et al., 1995).
The results of this study showed that totaland free testosterone levels tended to decrease
with aging, simultaneously with the penile NOconcentration and NOS expression. Testosterone replacement restored these levels. As mentioned above, testosterone may play a majorrole in the mediation of penile erection by affecting the pathway relaxing the corpus cavernosum smooth muscle in a manner that is dependent on the NOS activity. It is believed that

testosterone replacement might be an effective
method of treating erectile dysfunction that
occurs with aging.
In summary, the serum testosterone concentration, penile NO concentration and NOS expression decreased with aging and were restored by testosterone replacement. According to
the literature, NO plays a major role in the mediation of penile erection, and the present results
suggest that testosterone replacement might bean effective method of treating erectile dysfunction that occurs with aging.

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Received December 15, 2000; accepted January 12, 2001
Corresponding author: Dr. Manabu Shiono53