Effect of aging on expression of nitric oxide synthase I and activity of nitric oxide synthase in rat penis

Asian J Androl 2003 Jun; 5: 117-120             

Keywords: aging; nitric-oxide synthase; erectile dysfunction

Abstract

Aim: To investigate the effect of aging on the expression of nitric oxide synthase I (NOS I) and the activity of NOS in rat penis. Methods: Sixty male rats from 3 age groups (adult, old and senescent) were investigated. The expression of NOS I protein and mRNA in rat penis were detected by Western blot and RT-PCR respectively and the NOS activity, with ultraviolet spectrophotometry. Results: In the old and senescent group, NOS I protein expression was significantly decreased as compared with the adult. NOS I mRNA expression was well correlated with the protein expression. NOS activity was not statistically different between the adult and old groups, but it was significantly reduced in the senescent compared with the adult group (P<0.01). Conclusion: The aging-induced decreases in NOS I expression and NOS activity may be one of the main mechanisms leading to erectile dysfunction in the senescent rats.

1 Introduction

Erectile dysfunction (ED) is a universal problem in aging males and the occurrence is still increasing [1], but the causes leading to a decline in sexual function with age were not fully understood. Although geriatric diseases and the related medication may contribute to ED in the aged, the aging process itself undoubtedly plays a significant role.

A substantial body of evidence implicated nitric oxide (NO) in normal erectile function. The nerves regulating penile erection contain NOS I [2, 3]. NO donors and NOS inhibitors elicit and prevent erection, respectively [4, 5]. Penile erection begins with psychogenic stimuli as well as tactile stimuli that excite pelvic plexus neurons and activate the postganglionic cavernous neurons innervating the penis. Neural signals initiate penile erection by activating NOS I after depolarization-induced calcium entry and Ca2+/calmodulin stimulation of NOS I [6]. Neurally derived NO is well established as a mediator for penile smooth muscle relaxation, engorgement of cavernous sinusoids and subsequent erection [7]. NOS III, abundant in the endothelial lining of the penile vessels and trabecular meshwork, is also a potential source of NO [8]. Recent evidence had showed that NOS III was activated by the viscous drag/shear stress in blood vessels to produce NO continuously, a process that mediated the sustained (tumescence) phase of maximal penile erection [9]. Rajasekaran et al. [10] and Dail et al. [11] identified the expression and localization of these specific isoforms in the penile tissue. Gonzalez et al.[12] demonstrated the presence of NOS I gene and protein expression in the penis, however, the effect of aging on the protein and gene expression of NOS I were not defined.

The present work was designed to study the effect of aging on the protein and gene expression of NOS I in the rat penis.

2 Materials and methods

Three groups of male Sprague Dawley rats were employed in the study, including the Adult (5 months old, n=20), the Old (20 months old, n=20) and the Senescent groups (28 months~32 months old, n=20), categorized in accordance with the criteria of the National Institute of Health (NIH) classification on aging. Animals were maintained under standard conditions recommended by the NIH for 1 week before the experiment. They were then anesthetized and the shaft of the penis was carefully dissected and amputated at the level of the penile bulb. The penile shaft was snap-frozen in liquid nitrogen (-80 ) for further processing.

2.2 Preparation of protein extracts and western blot

Tissue extracts were prepared by homogenization in 10 volumes of ice-cold buffer A containing 20 mmol/L Tris-HCl (pH 7.5), 0.1 mmol/L Na₃VO₄, 25 mmol/L b-glycerophosphate, 2 mmol/L EDTA, 2 mmol/L EGTA, 1 mmol/L DTT, 1 mmol/L PMSF, 2 mg/L aprotinin and 2 mg/L leupeptin. After centrifugation at 16,000 g for 30 min, soluble protein was determined by using the BCA assay (Pierce, Rockford, USA). Equal amounts of protein (30 mg) were resolved on 7.5 % polyacrylamide gel and transferred to nitrocellulose membrane. Full Range Rainbow Molecular Weight Markers (Amersham, Piscataway, USA) were applied to one of the lanes. The resulting membranes were probed with anti-NOS I monoclonal antibody (1:500) (Santa Cruz Biotechnology, Santa Cruz, USA) and a secondary polyclonal IgG linked to horseradish peroxidase, followed by ECL system (Santa Cruz Biotechnology). Quantitation of protein bands was carried out by optical densitometry.

2.3 Reverse transcription-polymerase chain reactin (RT-PCR)

Semiquantitative RT-PCR was performed by determining the ratio of the products for NOS I and GAPDH; GAPDH was used as an internal standard in each tube to determine the baseline gene expression of that sample. Briefly, Total RNA was isolated from the fresh penile tissue (20 mg~100 mg) by the Trizol procedure (Gibco BRL, Gaithersburg, USA). The RT reaction was carried out on 1 mg RNA, applying 0.25 mg random primers, 0.1 mmol/L dNTP mixture, 40 units of RNasin ribonuclease inhibitor (Promega, Madison, USA), 200 units of Superscript II reverse transcriptase (Life Technologies, Grand Island, USA) in 1PCR buffer (10 mmol/L Tris-HCl, 1.5 mmol/L MgCl₂ and 50 mmol/L KCl, pH 8.3). The reaction was carried out at 37 for 1 h followed by 5 min of heating at 95 to destroy the enzyme and RNA. The resulting single-stranded cDNA (2 mL) was then subjected to 30 cycles of PCR under standard conditions. Samples were denatured at 94 for 3 min and after the addition of the polymerase, subjected to 30 cycles of amplification each consisting of 1 min at 94 , 1 min at 58 and 2 min at 72 , with a 10 min extension at 72 during the last cycle in the presence of 2.5 mmol/L Taq polymerase (Promega). The primers used to amplify NOS I corresponded to the following sequences: 5'-GAGACCTTCAACACCCCAGCC-3'(sense) and 5'-CTTTGGCCTGTCCGGTTCCC-3'(antisense). The rat GAPDH gene was also amplified as an internal control, using the primers: 5'-GTCGGTGTC-AACGGATTTG-3?(sense) and 5'-ACAAACATGGGG-GCATCAG-3'(antisense). The expected size of the amplicons was 213bp for NOS I and 397 bp for GAPDH. PCR products were separated by electrophoresis on 2 % agarose gels, stained with ethidium bromide and submitted to densitometry [13, 14].

NOS activity was measured by the methods previously described [15]. Briefly, tissues were homogenized in an incubation buffer containing 2 mmol/L leupeptin, 1 mmol/L pepstatin A and 1 mmol/L phenylmethylsulfonyl fluoride (PMSF). The cytosol fractions were incubated in triplicate for 15 min at 37 as indicated in the presence of 2 mmol/L NADPH, 100 mmol/L L-arginine and 0.45 mmol/L Ca²⁺. Absorbency was measured with ultraviolet spectrophotometry. The total NOS activity was calculated according to the Douglas methods and expressed as nmol/min/g tissue [16]. (NOS kit from Beijing Martial Academy of Medical Science, Beijing, China).

Data were expressed in meanSD. Student's t-test was employed to analyze the significance of difference. Statistical significance was established at P<0.05.

3 Results

Western blotting confirmed the presence of NOS I protein immunoreactivity at approximately 155 kDa in rat penile shaft. Optical densitometry indicated an age-related decrease in NOS I protein expression. The level of NOS I protein expression in the Old and the Senescent groups was 70.4 % and 61.2 % of that in the Adult, respectively. Significant differences were present between the Adult and Old groups (P<0.05) and the Adult and the Senescent groups (P<0.01) (Figure 1a, b).

Figure 1. Age-related expression of NOS I protein in rat penis. A: Luminol detection of the 155-kDa NOS I band. Lane 1: Adult; Lane 2: Old; 3: Senescent. B: Densitometric quantitation of band intensity (n=5). ^bP<0.05, ^cP<0.01, compared with the Adult group.

The same age-related decrease in NOS I mRNA expression was observed. The NOS I/GAPDH ratio was 0.490.05 in the Senescent, 0.610.06 in the Old and 0.710.07 in the Adult (Figure 2a, b). The result was in agreement with that of the NOS I protein expression.

Figure 2a, b. Age-related expression of NOS I mRNA in rat penis. A: nNOS and GAPDH bands appearing at 213 and 397 bp, respectively. M: Marker; Lane 1: Senescent; Lane 2: Old; Lane 3: Adult. B: NOS I/GADPH ratio (^bP<0.05, ^cP<0.01, compared with the Adult group).

The penile NOS activity (nmoles/min/g tissue) was 1.690.16 in the Adult, 1.820.23 in the Old and 1.190.18 in the Senescent. From Figure 3 it could be seen that the activity was similar between the Old and the Adult, while it was significantly lower in the Senescent than that in the Adult (P<0.01).

Figure 3. Effect of aging on NOS specific activity (n=20). ^cP<0.01, compared with the Adult group.

4 Discussion

Rats are generally considered to be resistant to naturally occurring and experimentally induced atherosclerosis [17], thus in the present study they were selected for the investigation of the pathophysiology of ED in aging.

Penile erection is elicited by the release of NO in penile nerve terminals as a result of central or peripheral sexual stimulation [18, 19]. This release occured mainly by the transient activation of NOS I [14] with the production of NO, which diffused to the adjacent target smooth muscle tissue and stimulates the guanylylcyclase activity. The subsequent elevation in cGMP triggers a reduction of cytoplasmic Ca²⁺ and the subsequent relaxation of corpora cavernosa. The present study indicated that in rats, aging was accompanied by a significant decrease of NOS I protein expression in the penis. The decrease was further supported by the finding that the NOS I mRNA was significantly less in old and senescent rats. A low NOS I level would lead to the decreased production of neurally derived penile NO. In accordance with our findings, Carrier and associates [20] demonstrated that the number of NOS-containing nerve fibers was significantly less in the old rats than that in the young.

The measurement of NOS activity in the penis may be taken as an indirect indicator of the potential NO synthesis. Our results also indicated that the penile NOS activity was significantly lower in the senescent rats than that in the other two groups. As a consequence, in the senescent rats the capacity of penile NO synthesis may be reduced with resultant impairment of the cavernous smooth muscle relaxation. A significant decrease in NOS activity together with the alterations in the collagen bundles of corpora cavernosa [21] may jointly harm the erectile response. This knowledge could have implications to the therapy of ED in the aged, since it had been shown that an improvement of the NO-dependent erectile response was feasible by either NOS induction or gene transfer [22].

It was anticipated that a balance of the NO level, the compliance of the smooth muscle and the release of contractile factors determines the rigidity and duration of erection. The possibility that aging increased the structural resistance of the corporal smooth muscle to relaxation would explain that an insignificant increase in penile NOS activity observed in the old rats was inadequate for achieving full rigidity.

In conclusion, our results indicate that aging causes a significant decrease of NOS I expression and NOS activity in the old rats, which may be one of the mechanisms leading to ED associated with aging.

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Correspondence to: Dr. Jun-Ping SHI, Department of Andrology, Shenyang Andrology Hospital, 32, Beiling Street, Shenyang 110032, China.
Tel/Fax: +86-24-2621 2469
E-mail: doctor.man@163.net
Received 2002-12-24   Accepted 2003-04-21

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