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a Department of Physiology, School of Medicine, University of La Laguna, 38071 Tenerife, Spain
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In addition to acting through specific neural circuits within the central nervous system (reviewed in [1]), androgens are now known to operate directly at the level of the penis and its autonomic innervation. Thus, in a widely studied animal model of penile erection, the intracavernosal pressure (ICP) response to electrostimulation of the pelvic nerves has been found to be considerably attenuated by castration or treatment with antiandrogens. This effect of androgen suppression is efficiently prevented or reversed by testosterone (T) or dihydrotestosterone (DHT) replacement [3–9].
An essential requirement for the development and maintenance of penile erection is the relaxation of the smooth muscle cells in the cavernosal trabeculae and their arteries. This phenomenon is physiologically regulated by a complex interplay of messenger molecules released from neural, paracrine, endocrine, and autocrine sources. The main neurochemical signal used by the erectogenic nerve fibers is thought to be nitric oxide (NO) (for reviews, see [10,11]), although other potentially important neurotransmitters cannot be excluded. The synthesis of NO is mediated by a family of three NO synthase (NOS) isoenzymes, encoded by different genes. That includes two "constitutive," Ca2+-dependent enzymes, originally described in neurons (nNOS) and endothelial cells (eNOS), both present in the cavernosal bodies [8, 12]. The third, Ca2+-independent, "inducible" isoform (iNOS) was first described in macrophages, although it has also been found in many other cell types, including corporal smooth muscle cells [13].
There is evidence that androgens can regulate NOS enzymes in the corporeal tissue. Thus, NOS activity in rat penis homogenates, as measured by the arginine/citrulline assay, has been found to decrease after castration [6–8, 14,15] and to be restored by T or DHT treatments [6, 7, 15]. Likewise, orchiectomy and androgen replacement have been reported, respectively, to decrease and to restore the NADPH-diaphorase staining (i.e., NOS activity) seen in nerve fibers within the cavernosal bodies [9].
However, there is controversy as to whether the castration-induced decrease in penile NOS activity is associated with a reduced expression of NOS proteins. Some groups have reported androgen-dependent changes in nNOS protein [6] and in the mRNAs of nNOS [15, 16] and eNOS [17]. Others found no effects of castration or androgen treatment on the levels of nNOS or eNOS proteins [8, 14,18] or eNOS mRNA [15].
Studies on the NO mediation of androgen actions on the penis have been limited by the lack of suitable methodologies for measuring physiological NO levels. Thus, the evidence currently available is derived essentially from either pharmacological studies in vivo, using drugs thought to act through NO mechanisms [2, 3, 5, 16, 19], or studies on the assessment of NOS activity in vitro [6, 7, 8, 14, 18].
Recently, we have developed an electrochemical approach for monitoring NO levels in the rat corpora cavernosa. The changes in the NO electrochemical signal can thus be recorded at intervals close to 1 min, concomitantly with those in ICP [20]. In the present work, this methodology was used to study the effects of castration and T replacement on the NO and ICP responses to cavernosal nerve stimulation. The changes in the NO response were compared with the content of the constitutive NOS isoforms in the corpora.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The experiments were done on Sprague-Dawley rats (250–300 g) reared at the University of La Laguna (Tenerife, Spain) Animal Facility. They were housed in a 12L:12D cycle with free access to rat chow and tap water. The experimental procedures complied with national regulations for the Care and Use of Laboratory Animals (similar to the NIH guidelines) and were approved by the local ethics committee. The experiments were conducted in accordance with the Guiding Principles for the Care and Use of Research Animals promulgated by the Society for the Study of Reproduction.
Animal Manipulation
Fourteen animals were orchiectomized under ether anesthesia and implanted s.c. with a Silastic capsule (Dow Corning, Midland, MI; o.d. 3.2 mm, i.d. 1.6 mm; 30 mm in length) that was either empty or filled with crystalline testosterone (Sigma Chemical Co., St. Louis, MO). An additional group of 8 animals was used as intact controls. Four weeks after these treatments, the animals were anesthetized with urethane (1 g/kg i.p.) and maintained at 37°C using a homeothermic blanket. To allow access to the corpora cavernosa and their nerves, the penile skin was removed and the abdomen was opened by a midline suprapubic incision. At the end of the electrochemical and pressure recordings, the animals were killed by an anesthetic overdose and their penises excised for NOS isoenzyme analysis.
Electrostimulation of the Cavernosal Nerve (SCN)
The major pelvic ganglion was identified at one side of the dorsolateral prostate, and a bipolar platinum electrode, connected to a Grass (Quincy, MA) S48 square wave stimulator, was placed on the emerging SCN. Nerve stimulation was performed for 1 min with 6-V, 12-Hz, 1-ms pulses.
Pressure and Voltammetric Recordings
The ICP was measured with a 23-gauge needle inserted into one corpus cavernosum, and the arterial pressure (AP) was monitored through a carotid line as previously described [20].
Voltammetry
Differential normal pulse voltammetry (DNPV) was performed with a carbon fiber-coated (30 µm in diameter x 500 µm in length) working electrode, mounted into a telescopic carrier assembly and inserted through the tunica albuginea [20]. The platinum auxiliary and Ag/AgCl reference electrodes were inserted into nearby abdominal muscles. Voltammetric recordings were made at 100-sec intervals with a microprocessor-controlled potentiostat system (Bioelectrochemical Analyzer, Tenerife, Spain). The DNPV parameters used were -100 to 1000 mV potential range, 20 mV/sec scan rate, 40-mV pulse amplitude, 40-ms pulse duration, and 50- to 120-ms prepulse duration. This results in a NO oxidation signal at approximately 650 mV that can be quantified. Details of the electrochemical procedure for measuring NO have been described elsewhere [20].
T Assay
Serum T levels were determined by ELISA using a commercial kit (DRG Instruments GmbH, Berlin, Germany) and an EL 311 (Bio-tek Instruments, Vermont, VA) spectrophotometer.
Western Blot Analysis of NOS Content
At the end of the electrochemical recordings, the corpora cavernosa were dissected and homogenized in SDS lysis buffer (0.075 M Tris-HCl, pH 6.8, 2.3% [w:v] SDS, 5% [w:v] ß-mercaptoethanol, and 10% [w:v] glycerol). After boiling for 5 min, the homogenates were centrifuged at 13 000 x g for 5 min, and the penile cytosols were recovered for gel analysis. An aliquot of each extract was preserved for protein quantification using a commercial DC protein assay kit (Bio-Rad, Hercules, CA). The remaining supernatant was diluted with an equal volume of double-strength Laemli loading buffer and frozen until time of electrophoresis.
Equivalent amounts of corpora cytosol protein (70 µg) were loaded and separated on 7.5% SDS-PAGE into a Mini-Protean II electrophoresis cell (Bio-Rad) according to the method of O'Farrell [21]. Proteins were electrophoretically transferred (16 h at 100 mA) onto Hybond-P PVDF membranes (Amersham, Arlington Heights, IL; now Amersham Pharmacia Biotech, Piscataway, NJ) using a Mini Trans-Blot electrophoretic transfer cell (Bio-Rad). The transfer efficiency was controlled by gel staining with Ponceau Red after electroblotting. Prestained protein molecular markers (Mr 210 000–46 000; Bio-Rad) were also run on each gel.
The immunodetection on the Western blots was carried out by first incubating for 3 h at room temperature with specific mouse anti-nNOS and anti-eNOS monoclonal antibodies (Transduction Laboratories, Lexington, KY) diluted 1:2000 in Blotto [22]. Membranes were washed (3 times, 20 min each) in PBT (PBS+0.05% Tween 20) and incubated for 3 h at room temperature with an anti-mouse IgG horseradish peroxidase-linked whole antibody (Bio-Rad) diluted 1:5000 in Blotto. After washing (3 times, 20 min each) again in PBT, NOS protein was visualized with the Amersham enhanced chemiluminescence kit.
X-OMAT x-ray films (Eastman Kodak, Rochester, NY) were exposed to luminol-reacted membranes and scanned with a Bio-Rad GS-670 densitometer. Each band density was evaluated with the Molecular Analyst software (Bio-Rad). Density values from the two experimental groups of rats were expressed as both densitometric units and percentage of the values for intact controls run on the same gel.
Data Processing
The data describing the NO responses were expressed as area under the curve (AUC), defined as time (20 min from onset of stimulation, during which 12 NO recordings were taken) x change (as percentage of basal level for NO recordings). The ICP and AP data were expressed, respectively, as the maximal ICP values found during nerve stimulation and the mean AP values recorded during that time. Data were analyzed by one-way ANOVA and post hoc Newman-Keuls tests. Linear regression analysis was used to compute best-fit lines and correlation coefficients. Statistical significance was set at P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Castration and T replacement also had effects on the basal NO electrochemical signals recorded in the corpora prior to SCN. The average current values recorded in the various groups were 1.21 ± 0.14 nA for I; 0.14 ± 0.04 nA for Cx (P < 0.001 vs. the other groups); and 1.20 ± 0.11 nA for CxT.
Both the ICP values and the NO signals were increased by SCN. However, these changes were minimal in the Cx animals (Figs. 1 and 2). T replacement (CxT group) increased the NO and the ICP responses to SCN, up to levels similar to those of the intact animals (Figs. 1 and 2). It is noteworthy that the mean AP values of the Cx males (74 ± 4 mm Hg) were significantly lower than in the other groups (I: 99 ± 4 mm Hg and CxT: 97 ± 5 mm Hg). Nonetheless, even if the ICP responses to SCN were expressed as the ICP/AP ratio, the above differences between groups still held at similar significance levels.
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Castration also decreased the levels of both nNOS and eNOS protein in the corporal homogenates, as measured by semiquantitative Western blotting; and T replacement efficiently prevented these effects (Fig. 3).
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To determine the relationship between the levels of NOS isoenzymes and the NO release in response to SCN, the three groups were pooled, and the NO response of each animal (expressed as AUC) was plotted against its penile content of nNOS, eNOS, or the combination of the two. Significant slopes and correlation coefficients were obtained with nNOS (r = 0.57, P < 0.01) and eNOS (r = 0.48, P < 0.05). The strongest correlation (r = 0.71, P = 0.0002) was observed with the values of nNOS and eNOS added together (Fig. 4).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The SCN-induced change in ICP was accompanied by a robust and longer-lived rise of the NO signals recorded in the corpora (Fig. 1). The possible reasons for this temporal dissociation between the NO and ICP responses have been discussed thoroughly elsewhere [20]. Several factors could account for this discrepancy. They include the transient quenching of NO by the blood filling the corpora during erection, the changes in cavernosal volume during erection and detumescence, and the relatively long distance from the NO source (nerve endings and endothelial cells) to the electrode tip placed in a large corporal volume. It is thus possible that the changes in NO levels detected at the electrode surface follow with some delay those actually occurring in the smooth muscle cells, presumably closer to the NO sources. An alternative possibility is that the NO levels reaching the cavernosal muscle cells remained, in fact, elevated for some time after the erectile response had subsided. That does not deny, however, the major role of NO in the regulation of penile erection. The contractile activity of the cavernosal smooth muscle depends on a complex balance of intercellular messengers derived from neural, endocrine, paracrine, and autocrine sources. Thus, the persistence of a poststimulation increase in NO levels outliving the ICP changes might well reflect the involvement of some additional, shorter-lived erectogenic signal(s). It is also possible that the relaxant action of NO is overridden by vasoconstrictor messengers (e.g., norepinephrine and/or peptides from neural and endothelial sources).
Both the NO and ICP responses to SCN were considerably attenuated in the castrated animals, their average values being, respectively, 20% and 15% of those of the intact rats, whereas T replacement fully prevented these changes. The observed effects of castration and T treatment on the ICP response to SCN are consistent with several published studies in the rat using a similar animal preparation and nerve stimulation protocol [4, 7, 8, 16]. The present data on corporal NO levels before and after SCN are also consistent with the postcastration decreases in NOS activity, as measured by the arginine/citrulline assay in penile homogenates, reported by other authors [6, 7, 14, 18].
However, it is worth noting that the effects of castration in the present work were more pronounced than in the studies cited above. The other groups all reported that both NOS activity and the ICP response to SCN were decreased after castration to around 50% of the levels in intact controls, whereas in this experiment, the corporal basal NO levels and the ICP and NO responses to SCN were well below that figure. Similarly, the observed effects of castration in nNOS and eNOS protein content (with these values falling, respectively, to 30% and 50% of those of the intact animals) were larger than in previous studies. Previous studies have usually shown milder postcastration decreases in nNOS [6] or no significant changes in these isoenzymes [8, 14, 18].
In addition to strain differences, a factor that could plausibly account for these discrepancies is the time elapsed since castration. Whereas in the other studies it was around 1 wk, in the present experiment we set a 4-wk postcastration interval before the in vivo recordings and tissue sample collection. The rationale was that several androgen-dependent measures of sexual behavior of the male rat take a relatively long time such as this to fade after castration. This is true not only for mounting behavior and other parameters of sexual motivation [1, 24] but also for some relevant indices of erectile activity. This phenomenon was described long ago in tests for erectile reflexes ex copula elicited in restrained supine rats by retraction of the penile sheath. Thus, some of these responses, especially those identified as "cups" and "flips" involving extrinsic androgen-dependent perineal muscles, fall in a few days after castration. However, the display of erections themselves continues to decline for several weeks [24, 25].
Few investigators have assessed the time course of the postcastration decline in the erectile responses to SCN as used in the present work. A study by Mills et al. [5] using Harlan-Holtzman rats showed a decrease of the ICP/AP ratio to about 50% of that for the intact controls 8 days after castration. By 5 wk later the erectile response was further reduced to nearly one third of the intact values, although the differences from the 8-day castrates were not statistically significant. In another study on Sprague-Dawley rats (the strain used in the present work), Zvara et al. [9] reported a gradual loss of the ICP response to SCN, with further significant decreases at various postcastration days ranging from 1 to 30. In fact, the minimal ICP response observed by these authors in their 30-day castrates was very similar to our findings. It is therefore likely that the long postcastration interval used in the present study allowed the effects of androgen deprivation on the erectile response to become clearer. That could also account for the smaller postcastration change, or the apparent lack of it, in nNOS or eNOS content reported in previous studies in which the tissue samples were taken 1 wk after surgery.
The strong correlation found in this study between the nNOS and eNOS levels versus the amount of NO released following nerve stimulation suggests that regulating the expression of these proteins is a main mechanism for the stimulatory actions of androgens in penile erection. Nonetheless, it is plausible that androgens can also influence constitutive NOS isoenzymes in the penis by mechanisms other than modulating their protein levels. This is supported by the previous studies on short-term castrates showing decreased NOS activity in spite of normal NOS protein content. Such findings suggest the involvement of androgen-dependent posttranslational phenomena.
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FOOTNOTES |
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2 Correspondence. FAX: 34 922 319397; mmas{at}ull.es
Accepted: May 17, 1999.
Received: March 9, 1999.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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