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Regular Article |
a Department of Urology, UCLA School of Medicine, Research and Education Institute, Harbor-UCLA Medical Center, Torrance, California 90509
ABSTRACT
Erectile dysfunction in the aging male results in part from the loss of compliance of the corpora cavernosal smooth muscle due to the progressive replacement of smooth muscle cells by collagen fibers. We have examined the hypothesis that a spontaneous local induction of inducible nitric oxide synthase (iNOS) expression and the subsequent peroxynitrite formation occurs in the penis during aging and that this process is accompanied by a stimulation of smooth muscle apoptosis and collagen deposition. The penile shaft and crura were excised from young (3–5 mo old) and old (24–30 mo old) rats, with or without perfusion with 4% formalin. Fresh tissue was used for iNOS and proteasome 2C mRNA determinations by reverse transcription polymerase chain reaction assay, ubiquitin mRNA by Northern blot, and iNOS protein by Western blot. Penile sections from perfused animals were embedded in paraffin and immunostained with antibodies against iNOS and nitrotyrosine, submitted to the TUNEL assay for apoptosis, or stained for collagen, followed by image analysis quantitation. A 4.1-fold increase in iNOS mRNA was observed in the old versus young tissues, paralleled by a 4.9-fold increase in iNOS protein. The proteolysis marker, ubiquitin, was increased 1.9-fold, whereas a related gene, proteasome 2c, was not significantly affected. iNOS immunostaining was increased 3.6-fold in the penile smooth muscle of the old rats as compared with the young rats. The peroxynitrite indicator nitrotyrosine was increased by 1.6-fold, accompanied by a 3.6-fold increase in apoptotic cells and a 2.0-fold increase in collagen fibers in the old penis. In conclusion, aging in the penis is accompanied by an induction of iNOS and peroxynitrite formation that may lead to the observed increase in apoptosis and proteolysis and may counteract a higher rate of collagen deposition in the old penis.
aging, apoptosis, male sexual function, nitric oxide, penis
INTRODUCTION
Penile erection is elicited by the release of nitric oxide (NO) in the nerve terminals of the penis as a result of sexual stimulation of central or peripheral origin [1, 2]. This release occurs mainly by the transient activation of the penile neuronal nitric oxide synthase variant (PnNOS) of the neuronal (n) NOS isoform [3, 4], producing NO, which diffuses to the adjacent target smooth muscle tissue stimulating guanylylcyclase. The subsequent elevation in cGMP triggers a reduction of cytoplasmic Ca2+ and the subsequent relaxation of the corpora cavernosa. The balance among the levels of NO synthesized in the penile nerves, the compliance of the smooth muscle, and the release of contractile factors determines the rigidity and duration of the erection. Erectile dysfunction that occurrs in rats with aging has been postulated to be caused by an alteration in this balance due to a reduced or insufficient synthesis of NO that is unable to compensate for the impaired cavernosal compliance resulting from an increase in collagen fiber deposition and a loss of smooth muscle cells [5]. There is experimental evidence for an age-related reduction in NOS activity and in the number of nitrergic nerve terminals in the corpora cavernosa [5, 6], accompanied by a decrease in the smooth muscle:interstitial collagen ratio [4, 7, 8], but no data are available on whether these processes are connected.
One of the factors in the corpora cavernosa that could affect simultaneously nNOS activity, penile nerve terminals, smooth muscle cell number, and collagen deposition is an abnormally high and persistent basal output of NO, independent of the erectile response itself. This type of NO release has been shown to occur under pathological conditions in several organs by the transcriptional stimulation of the expression of the inducible NOS (iNOS) [9, 10]. This enzyme is not subject to the regulation of enzyme activity operating for nNOS and endothelial NOS (eNOS) and is normally not expressed at detectable levels in the body. In contrast, nNOS and eNOS are physiologically maintained at a low basal level of activity, which is elevated intermittently during stimulation, and in general their level of expression is relatively constitutive [11]. Although iNOS induction is a well-known mechanism of immune defense triggered by cytokines against infection and cancer and during inflammation, it has recently been shown that this process may occur without an obvious underlying pathological condition in the aging brain and testis, leading to an elevated constant level of NO in the affected tissues [12–15].
The hyperproduction of NO as a result of iNOS induction or from the stimulation of nNOS activity by the N-methyl-D-aspartic acid receptor [12, 16], leads to the formation of peroxynitrite, a compound that can cause apoptosis and subsequent proteolysis, as shown in cell cultures and in several organs [10, 17, 18]. Peroxynitrite reacts with tyrosine residues in proteins to form nitrotyrosinilated derivatives [19], thus affecting their biological function. In the aging penis, this process could cause the loss of smooth muscle and nerve tissue involved in the erectile response. Conversely, an elevated synthesis of NO may be beneficial by having a paradoxical protective effect against apoptosis [17, 20] or by opposing fibrosis through the inhibition of collagen synthesis or proline hydroxylation [21–25]. In the present work, we investigated whether iNOS is induced spontaneously in the penile corpora cavernosa in the rat during aging and whether this induction is correlated with changes in peroxynitrite formation, apoptosis, proteolysis, and collagen deposition.
MATERIALS AND METHODS
Animals and Tissue Processing
Young (3 mo old) and old (22–24 mo old) male brown Norway rats were purchased from the NIH/NIA colony (Harlan Sprague-Dawley, San Diego, CA), maintained under controlled temperature and lighting, and treated according to NIH regulations. For certain experiments, Fisher 344 male rats from the same origin and of the same ages were used. Animals were anesthesized, and the penis was excised and denuded of skin. The glans and adhering noncrural tissue were removed, and the penile shaft and crura were stored at -80°C. For immunohistochemistry, anesthesized animals were pretreated with heparin and perfused through the left ventricle with saline followed by 4% formalin. The penile shaft was separated from the crura and fixed overnight in 4% formalin, washed with PBS, and stored in PBS at 4°C until further processing.
Determinations of Specific mRNA Levels in Total Penile Tissue
Total RNA was isolated from the combined fresh penile shaft and crural tissue by the Trizol procedure (Gibco BRL, Gaithersburg, MD), checking for the RNA quality by nondenaturing agarose gel electrophoresis/ethidium bromide staining [4, 26]. The reverse transcription (RT) polymerase chain reaction (PCR) assay was carried out on 1 µg of RNA, applying Superscript II RNase H- reverse transcriptase (Gibco BRL) and random primers (0.25 µg), followed by PCR with the respective gene-specific primers [4, 26]: 1) for iNOS, 22-mer primers at nucleotides 1590 (sense) and 1925 (antisense) of rat smooth muscle iNOS cDNA (GenBank number D14051), spanning an intron to exclude DNA contamination as the source of the expected 325-base pair (bp) band [27]; 2) for proteasome 2c, 20-mer primers at nucleotides 241 (sense) and 499 (antisense) of the rat proteasome 2c cDNA (GenBank number M29859), which generate a 258-bp band [26]. PCR products were separated by electrophoresis on 2% agarose gels, stained with ethidium bromide, and submitted to densitometry [4, 26].
Northern blot analysis for the expression of ubiquitin mRNA was performed on 20 µg total RNA [26] with a probe against maize ubiquitin mRNA with 100% homology to the rat ubiquitin mRNA (GenBank number 429159). Densitometry ratios were obtained between the intensities of the large (2.6 kilobase [kb]) or small (1.2 kb) ubiquitin bands, respectively, normalized by the intensity of the respective 28S band on the ethidium bromide-stained gel before transfer.
Determinations of iNOS Protein Levels in Total Penile Tissue
Tissue extracts were prepared from the fresh penile shaft/crura specimens by homogenization in 10 volumes of a medium containing 0.3 M sucrose, 0.02 M Hepes (pH 7.2), 1 mM dithiothreitol, and protease inhibitors, and the postmitochondrial supernatant was prepared [4]. Equal amounts of protein (50 µg) were run on 7.5% polyacrylamide gels and submitted to Western blot immunodetection with monoclonal anti-mouse iNOS (amino acids 772–787) IgG (1:500) and a secondary polyclonal horse anti-mouse IgG linked to horseradish peroxidase, followed by a luminol reaction [4, 26, 27]. The primary antibody detects the 130-kDa rat and human iNOS and does not cross-react with nNOS or eNOS. Both antibodies were from Transduction Laboratories (Lexington, KY).
Quantitative Determinations in Tissue Sections
The determinations of iNOS and nitrotyrosine [28] were carried out on 5-µm paraffin-embedded sections that were quenched for endogenous peroxidase activity after deparaffinization and rehydration, blocked with normal goat serum, and incubated with polyclonal anti-mouse iNOS IgG (1:500) (Transduction Laboratories) or polyclonal anti-nitroyrosine IgG (1:100) (Upstate, Lake Placid, NY). Negative controls omitted the first antibodies. The detection was based on a secondary anti-rabbit biotinylated antibody (1:200) (Calbiochem, La Jolla, CA), followed by the ABC complex (Calbiochem) and 3,3 diaminobenzidine (DAB) (Sigma, St. Louis, MO). Sections were counterstained with hematoxylin.
Inducible NOS and nitrotyrosine immunoreactivity were quantified by computarized densitometry using the Optimas program (Media Cybernetics, Silver Spring, MD) coupled to an Olympus BHS microscope equipped with a VCC video camera. Results were expressed as the inverse log of intensity gray value per cell area (ILIGV/AREA). In this program, after images are properly calibrated for background lighting, ILIGV/AREA results are proportional to the unweighted average optical density, which is used to determine the concentration of immunoreactive antigen.
TUNEL assays were done in the adjacent matched sections used for iNOS and nitrotyrosine staining [29] by applying the Apoptag Oncor kit (Oncor, Gaithersburg, MD) with terminal deoxynucleotidyl transferase (TdT) catalyzing the addition of digoxigenin-dUTP and -dATP to 3'-OH DNA fragment ends generated by internucleosomal cleavage. The resulting random heteropolymer of digoxygenin-dUTP and -dATP is detected with an antidigoxigenin antibody conjugated with peroxidase. Sections were treated with proteinase K, quenched with H2O2, incubated with digoxigenin-conjugated nucleotides and TdT, and then treated with antidigoxigenin-peroxidase. The apoptotic bodies were detected with DAB/H2O2, and sections were counterstained with methyl green. As a negative control, buffer was substituted for the TdT enzyme. Sections from testis of old animals were used as positive controls. The apoptotic cells were counted in a computerized grid, and the apoptotic index was expressed as the percentage of apoptotic cells within the total number of cells in the area (nonapoptotic nuclei plus apoptotic cells).
Collagen was estimated by histochemistry applying the Masson trichrome technique [30] followed by image analysis to measure the ratio between the width of the area stained positive for collagen (blue) divided by the total area of the penis.
At least 3 anatomically matched tissue sections per animal were analyzed in all quantitations.
Statistical Analysis
Values were expressed as mean ± SEM. The normality of the distribution of the data was established using the Wilks-Shapiro test, and the outcome measures between two groups were compared by the t-test. The difference among groups was considered significant at P < 0.05.
RESULTS
Physiological iNOS Induction in the Penis During Aging
To determine whether aging is associated in penile tissue with a spontaneous induction of iNOS, total RNA was isolated from the penile shaft and crura from 24-mo-old (old) and 3-mo-old (young) Brown Norway rats and submitted to RT-PCR with primers specific for rat iNOS. The amplified DNA was analyzed by agarose gel electrophoresis and ethidium bromide staining. Figure 1 (left panel, top) shows a distinctive 325-bp band of variable intensity for iNOS-amplified cDNA in the reaction for penile RNA from all the old animals, with a much fainter signal in one of the specimens. The iNOS-amplified band in the RNAs from the young penile tissue was less intense than that of four of five old animals. The densitometric analysis of the intensity of these amplified DNA bands (n = 5; Fig. 1, left panel, bottom) indicates a 4.1-fold higher level of iNOS mRNA in the old versus the young penile tissue.
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In another similar series of old and young rats, the postmitochondrial soluble protein extract was analyzed by Western blot for iNOS. A representative autoradiograph (Fig. 1, right panel, top) shows an intense 130-kDa band in the tissue supernatants from two old animals, which is absent in the extracts from two young rats and is similar to the iNOS band in the control extract from induced mouse macrophages. A smaller and fainter band of approximately 110 kDa was visible only in the penile extracts from the old animals. This band occurred as expected for the putative translation product of an iNOS mRNA variant detected in several tissues, which lacks deduced amino acids 289–427 [31]. Essentially the same results were obtained in the extracts from the other animals in each group (data not shown), and the densitometric analysis (n = 6) indicates a 4.9-fold stimulation of iNOS protein induction in the penis from the aged rats. This result is in agreement with the determination of iNOS mRNA levels. The same age-related increase in iNOS protein was observed in penile extracts from Fischer 344 rats (data not shown).
Cross-sections of the penis from young and old rats were then stained with a polyclonal anti-iNOS antibody adequate for immunohistochemistry. Figure 2, C and D, shows that iNOS staining in the old penile shaft is intense, diffuse along the corpora cavernosa and corpus spongiosum, and more concentrated in the endothelium of the cavernosal cisternae and blood vessels. In contrast, the penile shaft from the young rats (Fig. 2, A and B) shows little staining along these regions. A densitometric image analysis (n = 5; Fig. 2, E and F) showed that the mean number of immunoreactive cells was increased 3.6-fold in the old penile shaft as compared with the young shaft (Fig. 2E). The optical density per cell was also moderately increased (Fig. 2F).
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To confirm whether iNOS induction leads to a higher rate of NO and peroxynitrite synthesis, an antibody against nitrotyrosine was applied to cross-sections of penile shaft adjacent to the ones used for iNOS detection. Figure 3, A and B, shows that there is little staining in the penile sections from young rats, whereas it is considerably increased in the sections from the old animals (Fig. 3, C and D). This finding is reflected in a 1.6-fold increase in the number of immunoreactive cells (n = 5), with a slight increase in the staining intensity per cell (Fig. 3, E and F). The smooth muscle cells in blood vessels and in the cavernosal bodies were preferentially stained.
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Changes in Markers of Tissue Damage in the Penis During Aging
Because the overproduction of NO is linked to the induction of programmed cell death, sections adjacent to those examined above for iNOS and nitrotyrosine were stained with the TUNEL technique (n = 5). Figure 4, A and B, shows apoptotic cells in the penile shaft from the young rats, and the number of positive cells appears considerably increased in the old animals, to nearly 33% of the total cells in the examined areas. This is reflected in a 3.6-fold difference in the apoptotic index between the old and young penis (Fig. 4C).
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The age-related stimulation of the apoptotic index was reflected in a less intense change of proteolysis markers. As seen in Figure 5 (left), a cDNA probe specific for ubiquitin yielded a major 2.6-kb band and a minor 1.2-kb band for ubiquitin mRNA on a Northern blot. The densitometric ratio of the top band corrected by the 28S RNA is presented in Figure 5 (right), showing a 1.9-fold increase in the penile RNA from the old penises. The corresponding ratio for the small band (data not shown) was 2.3-fold.
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The age-related change in mRNA expression was much less marked with another component of the ubiquitin/proteasome pathway, the proteasome 2c mRNA (n = 5). Figure 6 (left) shows the ethidium bromide staining of the 258-bp DNA band generated by RT-PCR with primers for the rat gene and fractionated by agarose gel electrophoresis. The small increase induced by aging (14%) was not significant (Fig. 6, right).
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To determine whether the higher level of NO release in the old penile tissue was accompanied by changes in collagen deposition, adjacent tissue sections were also used for collagen detection utilizing the trichrome Masson stain. Collagen staining was much more intense in the penile shaft from the old than in that from the young rats (Fig. 7, A and B), as indicated by a 2.0-fold increase in the area occupied by collagen fibers (Fig. 7C).
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DISCUSSION
Our results indicate that aging in the rat penis is accompanied by a spontaneous induction of iNOS mRNA and protein and by the expected parallel increase in peroxynitrite formation as evidenced by the nitrotyrosinilation of proteins. This finding agrees with the higher number of apoptotic cells and ubiquitin expression in the aged penis and with an increased collagen deposition. These observations indicate that the augmented level of NO due to iNOS induction during aging would be deletereous to erectile function in the penis by causing apoptosis and subsequent proteolysis and by reducing the smooth muscle cell:collagen ratio in the penis. These changes would impair the compliance of the corpora cavernosa to the relaxation caused by NO released during sexual stimulation from nNOS-containing nerves. However, the well-established role of NO as an inhibitor of collagen deposition [21–25] suggests an antagonistic correlation between the increase in iNOS and collagen levels observed in the aging penis. In this case, iNOS induction may have a beneficial effect on erectile function by acting as a defense against the formation of more collagen fibers, in agreement with the dual roles exerted by NO on a series of connected physiological functions [10].
The age-related iNOS induction in the rat penis is substantiated by our findings of higher levels of iNOS mRNA by RT-PCR and of higher levels of iNOS protein by both Western blot and immunohistochemistry/image analysis. No inflammatory reaction was visible in tissue sections, indicating that infiltration by monocytes and related cells [32] was not the cause of the increased iNOS induction. The higher iNOS levels and peroxynitrite formation in the penis from old Brown Norway rats has also been documented in other tissues, such as the brain [12, 13, 15] and testis [14], and in the current work was also observed in an alternative animal model of aging, the Fischer 344 rat. The aged rats were in good health and were far from the end of their life span (32–36 mo). It is therefore unlikely that iNOS induction was triggered by an immune defense mechanism against a pathogenic infection or was the result of a generalized senile decay seriously disturbing homeostasis. It is more plausible that the observed age-related increase in tissue and circulating cytokines, such as tissue necrosis factor 
or interleukin ß-1 [12, 33, 34], is the main factor in the iNOS expression occurring in different organs of old rats. These biological effectors stimulate iNOS gene transcription through the activation of NF
B responsive elements in the iNOS promoter [9].
In both the testis and brain [12, 14], iNOS induction led to an increase of NOS activity in the tissue-soluble fraction measured by the L-argininine/citrulline assay, which agreed with the higher levels of peroxynitrite. In the current work, NOS activity was not measured in the penis because penile iNOS is not fully Ca2+ insensitive [35], impeding a good discrimination from nNOS and eNOS. Most of the NO pool originated from iNOS would lead to peroxynitrite formation, although a fraction may contribute to sustain smooth muscle relaxation during the erectile response, as observed upon gene therapy of the penis with iNOS constructs [27]. The increase in nitrotyrosine in the penile shaft from the old animals suggests that peroxynitrite arises mainly from iNOS induction; the levels of PnNOS [4] and eNOS (Gonzalez-Cadavid et al.) in the rat penis do not vary significantly with aging and may even decrease. Novel procedures to visualize basal in situ production of NO in tissues [36] and relate it to iNOS staining may be required for assigning peroxynitrite formation to iNOS containing cells because NO is a rapidly diffusing molecule.
The considerable increase of apoptotic cells in the old rat penile corpora cavernosa supports the concept that aging is accompanied by a relative reduction of smooth muscle tissue in the penile shaft and that this reduction may be related to the increased peroxynitrite formation. The age-related loss of penile smooth muscle has been reported in the rat [5, 6, 8] and human [7], and it is assumed to contribute to the gradual loss of cavernosal compliance. The apoptotic index was surprisingly high in the middle shaft of the aged penis, and even in the young penis, above what could be expected from the replacement of smooth muscle cells by collagen fibers in an organ that does not experience a substantial loss of mass with aging. However, our data are consistent with those from studies showing a considerable level of programmed cell death in the penis by methods applied to tissue extracts [37, 38]. These findings suggest that smooth muscle cell proliferation continues to occur at a high rate and compensates for the cells eliminated by apoptosis by a sort of tissue remodeling [39] superimposed on the net decrease in the smooth muscle:collagen muscle ratio.
The induction of apoptosis by peroxynitrite is well documented in cell cultures and in vivo in animal tissues [17–20], although it has not been reported in the penis. The increase of iNOS and nitrotyrosine in the penis is accompanied by a higher apoptotic index, which in turn coincides with an elevated expression of a proteolytic marker, ubiquitin. The stimulation of the ubiquitin-proteasome pathway is associated with the loss of skeletal muscle induced by catabolic agents and sepsis [26]. However, a direct demonstration of a causal relationship between iNOS induction/higher peroxynitrite formation and the stimulation of cavernosal smooth muscle apoptosis and an examination of their relationship to the impairment of cavernosal relaxation observed in old rats [5] is needed. The increase in NO may be preventive rather than deletereous, aimed at destroying reactive oxygen species (ROS). These compounds are inducers of apoptosis, and their accumulation is linked to the aging process [40, 41]. Low concentrations of NO appear to protect cells from apoptotic cell death [17, 20, 42] so that the levels of NO, as well as their balance with ROS levels, would ultimately dictate the degree of cell damage, thus confirming the dual role of NO [10].
The parallel increases of iNOS and collagen synthesis in the rat penis with aging, observed also in experimental fibrosis of the penile tunica albuginea in adult rats [43], may arise from an antagonistic rather than agonistic interaction between these processes. Inducible NOS induction and the increase of NO synthesis may be a defense mechanism against collagen deposition and fibrosis, in agreement with the inhibitory effects of NO on collagen synthesis and the development of fibrosis in several organs by chronic NOS blockade [21–25]. Physiological iNOS induction during aging may then be the body's defense mechanism to slow down an accelerated rate of collagen formation induced by endogenous fibrotic factors.
This study produced the first demonstration of iNOS induction and nitrotyrosine accumulation in the penile shaft with aging and the association of these processes with apoptosis and collagen deposition. To determine whether this increased NO output is truly cytotoxic and irrelevant to fibrosis or whether it is a preventive physiological mechanism where the damaging effects to cells coexist with the inhibition of more collagen synthesis, a functional demonstration is needed. Even the question of whether NO is a factor opposing apoptosis in the aging penis remains open. The study of these end points in iNOS knockout mice [19] during aging may help to elucidate these interactions, although transgenic animals develop compensatory pathways that may complicate the interpretation. The use of new selective inhibitors of iNOS enzyme activity or expression [44–46] and long-term NO donors [47] in wild-type strains may provide more conclusive information.
ACKNOWLEDGMENTS
The collaboration of C. Hayden in immunohistochemical detection is gratefully acknowledged.
FOOTNOTES
First decision: 26 September 2000.
1 This work was supported by NIH grant R01DK53069 (N.F.G.-C.). 
2 Correspondence: Nestor Gonzalez-Cadavid, Harbor-UCLA Medical Center, Department of Surgery, Division of Urology, Building F-6, 1000 West Carson St., Torrance, CA 90509. FAX: 310 222 1914; ncadavid{at}ucla.edu
Accepted: October 31, 2000.
Received: August 3, 2000.
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