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Male Reproductive Tract |
a Department of Urology, UCLA School of Medicine, Los Angeles, California 90095
b Research and Education Institute, Harbor-UCLA Medical Center, Torrance, California 90502
c Johns Hopkins University, Department of Urology, Baltimore, Maryland 21287
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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aging, gene regulation, male reproductive tract, nitric oxide, penis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The main nNOS variant in the penis, presumably responsible for nitrergic neurotransmission during sexual stimulation, differs from the brain-type nNOS variant in that a 34-amino-acid insert is present in the penile variant between the sequences encoded by exons 16 and 17, and this been named penile nNOS (PnNOS; also referred to as nNOSmu in skeletal muscle) [8, 11–13]. The control of enzyme activity in different nNOS variants is essentially exerted through the binding of Ca2+ to calmodulin. The increase of Ca2+ levels in the postsynaptic neuron cytosol occurs via the activation of N-methyl-D-aspartate receptors (NMDAR) by excitatory amino acids [5, 6, 14, 15]. In addition, NMDAR activity is coupled to nNOS through the binding of a protein, PSD95, to the nNOS PDZ domain encoded by exon 2. In the rat penis, NMDAR subunits and putatively active receptors have been detected [15].
Additional control pathways for PnNOS may occur by the binding of two proposed physiological inhibitors of nNOS activity: CAPON (carboxy-terminal PDZ ligand of nNOS) and PIN (protein inhibitor of NOS). The CAPON interacts with the PDZ domain competing with PSD95, and PIN binds in a close region further downstream. The PIN is a 89-amino-acid protein that acts on nNOS by impeding its dimerization [16–18]. It has been detected in the brain [16, 19], specifically in the hypothalamus in regions involved in the control of penile erection, colocalizing in certain neurons with PnNOS [20]. However, the role of PIN in erectile function is unclear, because in the hypothalamus, it exhibited marginal inhibitory activity [20] in comparison to strong inhibition during in vitro studies with recombinant nNOS [16, 21, 22]. In addition, it is also expressed in nonneural tissues [23, 24]. Because PIN is homologous to the light-chain dynein [18, 25–29], a component of microtubules, it has been proposed that PIN may also be involved in nNOS association with the neuronal cytoskeleton during axonal transport [25, 26]. Truncated protein splice variants of both PnNOS and the brain-type nNOS arise from alternative splicing that bypasses exon 2 [8, 30], and conceptually, they should be refractory to modulation by PIN, CAPON [31], and NMDAR. These nNOS variants are named ß forms to differentiate them from the full-length 
forms. The ß forms have been detected in both rat and mouse penile nerves [8], and their persistence in the nNOS knockout mouse may explain the unexpected normal erectile function in this animal.
In the present study, we have extended our previous reports on PnNOS and NMDAR expression in the rat and mouse penis [8, 11, 12, 15] by determining whether PIN and NMDAR are both expressed in penile nerves and whether they colocalize in neurons with PnNOS and persist in the nNOS knockout mouse. In addition, we have determined whether the mRNA expression of PnNOS, brain-type nNOS, PIN, and NMDAR in the rat penis are affected by aging and whether PIN can actually bind to penile nNOS and affect its activity in vitro in rat penile homogenates or impair erectile function in vivo.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Young (age, 5 mo) and old (age, 24 mo) male Fisher 344 rats were obtained from the National Institutes of Health/National Institutes of Aging colony (Harlan Sprague-Dawley, Inc., San Diego, CA) and maintained under controlled temperature and lighting according to NIH regulations. Young (age, 3 mo) nNOS-/- and nNOSwt mouse strains were as described previously [8] and were bred and maintained at the Johns Hopkins animal facility. Animals were anesthetized with thiopental (25 mg/kg body weight), pretreated with heparin, and perfused through the left ventricle with saline followed by 4% formalin [32]. The whole penis was removed, denuded, postfixed overnight in 4% (w/v) formalin, washed with PBS, and stored in PBS at 4°C until further processing within the first 24 h. For cryostat sectioning, rat and mouse tissues were treated with OCT embedding compound (Sakura Finetek, Torrance, CA) and frozen at -80°C. Additional young and old rats were used for dissection of the skin-denuded penile shaft and crura (eliminating the glans) and, in certain cases, for dissection of the cavernosal nerve and pelvic ganglion, without prior heparinization and perfusion with fixative. Particular care was applied to avoid the presence of perineal striated muscles adjacent to the crura. Testis, skeletal muscle, heart, and prostate were also excised from three rats. All fresh tissues were immediately frozen in liquid nitrogen and stored at -80°C. The experimental procedure was approved by the Research and Education Institute's Animal Care and Use Review Committee.
Detection of PIN mRNA in Tissues
Total RNA was isolated from the nonfixed tissue by the Trizol procedure (Gibco BRL, Gaithersburg, MD) and submitted (1 µg) to reverse transcription (RT) using Superscript II RNase H- reverse transcriptase (Gibco BRL) and random primers (0.25 µg), followed by polymerase chain reaction (PCR) with the respective gene-specific 20-mer primers spanning an intron to exclude DNA-based PCR contamination [8, 15, 20, 32]. The number of cycles and the concentration of primers were adjusted to maintain amplification of each PCR product within the linear range. Primers used for PCR were as follows: for nNOS brain variant and PnNOS, on nucleotides (nt) 2561–2580 (forward) and 2788–2807 (reverse) of the rat PnNOS cDNA (GenBank no. U67309), as the source of the expected 247-base pair (bp; PnNOS) and 145-bp (nNOS, brain variant) bands; for PIN, F1 on nt 74–93 (forward) and R1 on nt 401–420 (reverse) of the rat PIN cDNA (GenBank no. U66461), as the source of the expected 347-bp band, as well as F2 on nt 84–103 (forward) and R2 on nt 349–368 (reverse), generating a 295-bp band, and F3 on nt 99–118 (forward) and R2 (reverse), generating a 270-bp band; for NMDAR-1, nt 2522–2541 (forward) and nt 2785–2804 (reverse) of the rat NMDAR-1 cDNA (GenBank no. X63255), as the source of the expected 283-bp (NMDAR1, brain variant) and 334-bp bands (NMDAR1-T, penile variant). The PCR products were separated by electrophoresis on 1.5% agarose gels and stained with ethidium bromide. For densitometry, normalization was performed against the ß-actin (rat ß-actin control amplifier set; Clontech, Palo Alto, CA) housekeeping gene fragment generated in the same PCR reaction, giving a band of 764 bp. Total RNA was also analyzed for PIN mRNA expression by Northern blot on formaldehyde 1% agarose gels using hybridization with a cDNA probe corresponding to the 347-bp, full-length rat PIN cDNA labeled with [32P]dCTP by random priming [20].
Cloning of Penile PIN cDNA
A construct of the full-length rat PIN cDNA was prepared by PCR amplifying the PIN cDNA from a rat corpora cavernosa lambda library [11]. The PCR primers were based on the sequence for the brain PIN cDNA (nt 74–93, forward; nt 401–420, reverse; GenBank no. U66461 [16]) and flanked the PIN coding region. The PIN cDNA fragment was gel purified using a Qiaquick gel extraction kit (Qiagen, Valencia, CA) and blunt-end ligated into the bacterial cloning vector pCRScriptAMP (Stratagene, La Jolla, CA). Both forward- and reverse-orientation clones were identified by DNA restriction digestion and DNA sequencing. The PIN cDNAs were then subcloned in both orientations into the mammalian expression vector pCDNA3 by excising the PIN fragment with NotI and XhoI restriction enzymes and ligating into pCDNA3 between the NotI and XhoI restriction sites. The sense construct, referred to as pCMV-PIN, was also sequenced.
Immunohistochemical Detection
Tissue detection of PnNOS, nNOS, PIN, and NMDAR was carried out on sections (thickness, 6 µm) obtained with a cryostat from the OCT frozen tissue and fixed for 15 min on Color-Frost Plus microscope slides (Fisher Scientific, Hampton, NH)with 4% paraformaldehyde in Soresnsen solution (0.1 M phosphate buffer) [11]. Sections were washed with PBS, permeabilized with 0.4% Triton X-100, blocked with 1.5% normal goat serum in PBS, and incubated overnight in a humidified chamber at 4°C with the following primary antibodies: anti-rat PIN, described above (1:500 [w/v]); b) anti-PnNOS (1:500 [w/v]) polyclonal immunoglobulin (Ig) G (custom made; Bethyl Laboratories, Montgomery, TX), directed against 16 amino acids in the 34-amino-acid insert [8]; anti-rat NMDAR 2 subunit (1:300 [w/v]) monoclonal IgG (BC Pharmingen Transduction Laboratories, San Diego, CA); and anti-human synaptophysin (1:100 [w/v]; Zymed Laboratories, San Francisco, CA) [8]. Biotinylated goat anti-rabbit secondary antibody (1:200 [w/v]) was applied and incubated for 30 min, followed by 30 min with avidin-biotin-horseradish peroxidase complex. Antibody binding was visualized with diaminobenzidine solution, and sections were dehydrated and cover slips mounted.
In other instances, the detection was carried out on paraffin-embedded penile sections (thickness, 5 µm), collected onto gelatin-coated slides [20, 33]. Sections were quenched for endogenous peroxidase activity after deparaffinization and rehydration, blocked with normal goat serum or normal horse serum, and incubated with the antibodies listed above, except that PIN was used at 1:300 (w/v). Negative controls were done by replacing the first antibody with nonimmune IgG. Each slide assayed had a negative control omitting the first antibody. Sections were counterstained with hematoxylin. In the case of the monoclonal nNOS antibody (BD Pharmingen Transduction Laboratories [w/v]), the secondary antibody was an anti-mouse biotinylated IgG (1:200 [w/v]; Vector Laboratories, Burlingame, CA).
Double-Labeling Immunodetection
Colocalization of PnNOS neurons with PIN and with NMDAR subunit 2B was performed on paraffin sections, placed onto gelatin-coated slides. After deparaffinization and hydration, sections were preincubated with 10% goat serum or horse serum and then in a 1:100 dilution of the anti-PIN polyclonal antibody or a 1:300 dilution of anti-NMDAR2B monoclonal antibody, followed by a 1:20 dilution of anti-rabbit biotinylated secondary antibody or anti-mouse biotinylated secondary antibody (Vector Laboratories). Sections were then incubated in 20 µg/ml of streptavidin-fluorescein isothiocyanate (FITC; Vector Laboratories), followed by 10% goat serum and then a 1:500 dilution of anti-PnNOS or anti-nNOS antibody. Fluorescence labeling was performed with anti-mouse or anti-rabbit secondary antibody-Texas red (13 µg/ml; Vector Laboratories). Sections mounted in Prolong Anti-Fade (Molecular Probes, Eugene, OR) were examined using a Leica TCS SP confocal laser scanning microscope (Leica Microsystems, Buffalo, NY) equipped with argon and HeNe lasers.
Test of PIN on NOS Activity in Tissue Homogenates
The NOS activity was measured by a modification of methods previously described [9, 10]. Briefly, penile and cerebellar tissues were homogenized in a 1:6 (w/v) ratio in a buffer containing 0.32 M sucrose, 20 mM Hepes (pH 7.2), 0.5 mM EDTA, 1 mM dithiothreitol, and protease inhibitors (3 µM leupeptin, 1 µM pepstatin A, and 1 mM phenylmethyl sulfonyl fluoride). The particulate and cytosolic fractions were obtained by centrifugation at 12 000 x g for 60 min, and the soluble fraction was passed through Dowex AG50WX-8 (Na+) resin (Sigma Chemical Co., St. Louis, MO) to remove endogenous arginine. Samples were then incubated at 37°C for 30 min with L-[2,3,4,5-3H]arginine monohydrochloride (63 µCi/nmol; final concentration, 3 µCi/ml; Amersham Biosciences, Piscataway, NJ), 2 mM NADPH, 0.1 mM L-arginine, and 0.45 mM calcium. Controls included the addition of a NOS inhibitor (N
-nitro-L-arginine, 2 mM), a Ca2+ chelator (EGTA, 5 mM), or the omission of Ca2+. Recombinant PIN-GST (glutathione S-transferase) was prepared as described previously [20] and incubated for 30 min at 0°C before determination of NOS activity. At completion, the residual L-[3H]arginine was eliminated through the resin, and [3H]citrulline was counted in the trichloroacetic acid/ether-extracted supernatant. Background activity was corrected by subtracting for time-zero incubations. The NOS activity is expressed as picomoles of L-citrulline per minute per milligram of protein.
PIN/PnNOS Protein Interaction
The GST-PIN and GST proteins (20 µg each in 100 µl of PBS) were incubated with tissue extracts from rat hypothalamus and cerebellum (100 µl each) for 1 h at 4°C. All subsequent procedures were done at 4°C. The reactions were passed over glutathione-Sepharose 4B columns (bed volume, 200 µl; Amersham Biosciences) that bind GST, washed extensively with HNTG buffer (20 mM Hepes [pH 7.4], 500 mM NaCl, 10% glycerol, and 0.1% Triton X-100), and eluted with three 400-µl aliquots of glutathione elution buffer (100 mM Tris [pH 8.0] and 10 mM glutathione) [16, 20]. One-tenth of each eluted aliquot was run on 4–20% gradient polyacrylamide gels and submitted to Western blot immunodetection with a monoclonal anti-rat PIN (whole length, 89 amino acids) IgG at 1:1000 dilution (BD Transduction Laboratories, Lexington, KY), followed by an anti-mouse goat IgG linked to horseradish peroxidase (BD Transduction Laboratories) or by the anti-nNOS antibody used above for immunohistochemistry, followed by the anti-rabbit IgG linked to peroxidase. Visualization of the bands was performed by a luminol reaction (Pierce Endogen, Rockford, IL). Negative controls were performed without primary antibody.
Measurement of Erectile Response to Electrical Field Stimulation
Aged rats (24 mo) were used for examining the erectile-promoting effects of PnNOSß cDNA, and adult rats (5 mo) were used for experiments involving PIN cDNA. Animals were anesthetized with ketamine:xylazine mix (70:7 mg/kg body weight), the penis exposed, and a ligature placed at the base of the penis. The animals were injected in the corpora cavernosa in the middle of the penile shaft with 100 µl of either saline alone or the appropriate cDNA construct in saline, as indicated in each experiment, and the ligature was removed 2 min later. Immediately following injection, electroporation was applied. It was given with 0.5-cm platinum electrodes spanning the site of injection longitudinally and utilizing the Electro Square Porator ECM 830 (BTX, San Diego, CA). Settings were 100 V (voltage), 40 msec (duration), 8 pulses/sec (frequency), 1 sec (interval), and unipolar (polarity), as optimized in preliminary experiments and adapted from a protocol devised for the rat skeletal muscle [12].
At the indicated periods, animals were used for determinations of erectile response to electrical field stimulation (EFS) as described previously [9, 12]. Briefly, after induction of general anesthesia, a midline surgical approach was used to obtain exposure of the cavernosal nerve. Platinum electrodes were applied to the cavernous nerve, and arterial and intracavernosal pressure measurements were obtained by simultaneous direct intrafemoral artery and cavernosal catheterization, respectively. The EFS was applied at 10 V and a frequency of 15 Hz for pulses of 30 sec, separated for 2-min intervals, with a Grass Stimulator (Grass Instruments, Quincy, MA). A data acquisition system (Biopac Systems, Santa Barbara, CA) connected to a personal computer simultaneously recorded arterial blood pressure (femoral artery) and intracavernosal pressure, and values were expressed as mm Hg (mean ± SEM). The ratios between the maximal intracavernosal pressure (MIP) and the mean arterial pressure obtained at the peak of erectile response (MAP) were calculated to normalize for variations in blood pressure.
Statistical Analysis
Values were expressed as the mean ± SEM. The normality distribution of the data was established using the Wilk-Shapiro test, and the outcome measures between two groups were compared by the Student t-test. Differences between two groups were considered to be significant at P < 0.05. Multiple comparisons among the different groups were analyzed by a single-factor analysis of variance, followed by post-hoc comparisons with the Student-Newman-Keuls test, according to the GraphPad Prism version 3.0 program (San Diego, CA). A level of P < 0.05 was considered to be significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In previous studies, we had found that both PnNOS and NMDAR mRNA and protein were expressed in the rat penile shaft [8, 15], and in the present study, a similar approach was conducted for PIN. Utilizing three sets of primers derived from the sequence of a clone of rat penis PIN cDNA [20], the expected 270-, 295-, and 347-bp fragments were generated by RT-PCR from RNA isolated from the penile shaft as well as from the skeletal muscle, heart, prostate, and testis, thus showing that PIN mRNA was present in the penis as expected but also expressed ubiquitously (Fig. 1, top). Within the penis, the localization of PIN mRNA was seen specifically in the cavernosal nerve as well as in the pelvic ganglion (Fig. 1, bottom left). Northern blot analysis (Fig. 1, bottom right) confirmed PIN mRNA and protein expression in the penile shaft. A possible longer mRNA transcript is also present.
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A cDNA library was obtained for rat penis and screened using the PIN DNA probe applied to Northern blots, and one of the cDNA clones was sequenced. The resulting sequence was 100% identical to that of PIN cDNA cloned from rat brain [16]. This DNA fragment, subcloned into an eukaryotic expression vector, was able to express the PIN 10-kDa protein in 293 cells, as detected by Western blot analysis with a polyclonal custom-made antibody [20]. This showed that the PIN mRNA identified in the rat penis by RT-PCR and Northern blot analysis does, in fact, encode the expected 89-amino-acid PIN protein and that the antibody used below for immunohistochemistry is immunoreactive with the recombinant PIN protein produced in transfected 293 cells.
The immunodetection of PIN and the neuronal marker synaptophysin was carried out in frozen serial cross-sections of the rat penile shaft. Figure 2 shows at high magnification the outer ventral part of the shaft with skin and fascia removed, focusing on a nerve bundle and other nerve endings that stain positive for both PnNOS (Fig. 2, top left) and synaptophysin (Fig. 2, bottom right). The PIN was detected in what appears to be the same neural structures (Fig. 2, top right) and in scattered cells of the corpora cavernosa, with some appearing to be either nerve terminals, as shown by synaptophysin costaining, and others possibly being smooth muscle. Staining for the NMDAR subunit 2B is evident in the same region but scarce and much less intense than PIN (Fig. 2, bottom left).
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The determination of colocalization of PIN and PnNOS in penile nerve bundles was confirmed by repeating, in paraffin-embedded sections, PIN staining (Fig. 3, bottom left) colocalized with PnNOS (Fig. 3, top and left). The PIN is seen in some structures of the corpora cavernosa that appear to be PnNOS-expressing nerve endings and in many nonneural cells (Fig. 3, bottom right), in which scattered PnNOS staining was also observed (Fig. 3, top right). A similar finding is observed with NMDAR subunit 2B, in which the immunoreactivity is more evident than in Figure 2 and is clearly present in branches of the cavernosal nerve as well as in what appears to be some smooth muscle cells (Fig. 4, top left). Localization of NMDAR in the cavernosal nerve is confirmed at higher magnification (Fig. 4, bottom right), which strikingly shows the absence of NMDAR in the dorsal nerve (Fig. 4, bottom left). A distinct pattern of NMDAR staining is seen in smooth muscle cells around cavernosal cisternae (Fig. 4, top right).
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Colocalization of PnNOS and Associated Proteins and Effect of Aging on Their Expression in the Rat Penile Shaft
The definitive confirmation that both PnNOS regulatory proteins, PIN and NMDAR, were expressed in nNOS- or PnNOS-containing nerves was obtained by dual-fluorescence staining of sections showing branches of the dorsal and cavernosal nerves. Nerve terminals expressing PIN and NMDAR were separately labeled with the respective antibodies attached to a green fluorescence FITC tag (Fig. 5, top left and top right, respectively), and the nNOS- or PnNOS-positive cells were stained with the respective antibodies linked to red fluorescence (Fig. 5, middle). The nNOS antibody was used for PIN colocalization instead of the PnNOS antibody because both PnNOS and PIN antibodies are raised in the same species (polyclonal). The overlay shows, in yellow, that a number of nNOS axons (nerve terminals) in the dorsal nerve express PIN (Fig. 5, bottom left), particularly in one definite area of the nerve, whereas other regions do not have this colocalization. In contrast, the NMDAR fluorescence staining was negative in the dorsal nerve (Fig. 5, top right), as was seen in Figure 4, and this absence of NMDAR was confirmed in a number of sections examined. In the case of the cavernosal nerve, both PnNOS and NMDAR expression were clearly seen (Fig. 6, top left and middle left, respectively), and both proteins colocalized in the overlay (Fig. 6, bottom left). Interestingly, some cavernosal nerve branches that express NMDAR (Fig. 6, top right) do not express PnNOS (Fig. 6, middle right and bottom right).
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Aging in the rat is accompanied by a decrease in NOS activity in the penis [7, 9] and impairment of cavernosal smooth muscle relaxation [7, 9]. We investigated the possibility that this results from a decrease in PnNOS or its positive modulator, NMDAR, or an increase in its negative modulator, PIN. The levels of the respective mRNAs were estimated by semiquantitative RT-PCR, and Figure 7 (left) shows that no significant change occurred with aging in PnNOS mRNA. Utilizing a set of primers that also detects the brain-type nNOS as a smaller fragment, relatively little mRNA expression was observed for this variant in the penis, and no changes in nNOS were seen with aging. In the case of PIN (Fig. 7, middle), the levels remained constant in old compared to adult rats, and the same occurred with NMDAR1, represented in the penis by the truncated mRNA variant, NMDAR1-T (Fig. 7, right).
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Persistence of Expression of PIN and NMDAR in the nNOS Knockout Mouse
In a previous study, we have shown that the truncated ß form of PnNOS is expressed in the nNOS knockout mouse, both as RNA and protein, despite the disappearance of the full-length form and that this result may explain the presence of erectile function in this knockout mouse [8]. When examining nNOS modulators in frozen cross-sections of the penile shaft of the wild-type mouse, PIN can be detected (Fig. 8), and this expression persists in the nNOS-/- mutant. The synaptophysin staining (not shown) in this mouse model is consistent with expression occurring in nerve terminals, as is seen in the rat, but PIN is more ubiquitously expressed within the corpora cavernosa than synaptophysin. Staining for the NMDAR subunit 2B was also visible in nerve endings in the mouse, but as in the rat, the intensity was not high (not shown).
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Effects of PIN on Penile NOS and Erectile Function
To determine whether PIN has the capacity to inhibit penile NOS activity, pure recombinant PIN-GST protein (20 µg) was added to penile cytosol extracts containing nNOS (50 µl) and incubated for 60 min, and the L-arginine/citrulline conversion assay was performed to assess NOS activity. No significant effect of PIN on NOS activity was observed in the supernatant, and the same situation occurred with detergent 3-[(3-cholamidopropyl)dimethylammonio]-1propanesulfonate extracts of the membrane pellet (15 000 x g, 60 min; data not shown). The GST protein control had no effect, as previously shown [16].
Another approach to assess the functional role of PIN was also performed in vitro. The ability of PIN to interact with penile nNOS was investigated by binding experiments in which recombinant PIN-GST was incubated with cytosolic and pellet extracts from separate homogenizations of the penile shaft and crura and then passed through a GST-Sepharose 4B column to retain the PIN-GST protein. As a negative control, tissue extracts were also incubated with GST and run over GST-Sepharose 4B columns. A cerebellar supernatant was used as a positive control in similar incubations. After thorough washing, proteins bound to the columns were eluted and subjected to electrophoresis under denaturing conditions on PAGE gels followed by Western blot analysis. Both nNOS and PIN antibodies were used for immunodetection. Figure 9 (bottom left) shows that nNOS from the cerebellar supernatant was retained by GST-PIN bound to the glutathione-Sepharose 4B column but not by GST alone, indicating a specific interaction. This is evidenced by the immunodetection of nNOS in the fractions retained by the PIN-binding column and eluted. In contrast, no such interaction between nNOS and PIN occurred in the penile extracts, as shown by the absence of the nNOS band despite nNOS being present to some extent in the flow-through fraction (Fig. 9, bottom right). The PIN-GST was eluted as expected (Fig. 9, left top), indicating that the failure to detect nNOS in this fraction was not caused by PIN retention in the column.
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The ultimate assessment of PIN biological activity on the nitrergic pathway controlling penile erection, however, is determination of the effects exerted by PIN on the erectile response to EFS of the cavernosal nerve. Figure 10 (left) shows that when a plasmid construct of PIN cDNA (30 and 100 µg) was given by injection and electroporation [12] to the corpora cavernosa of adult rats (age, 5 mo) and the MIP:MAP ratio during EFS was measured at 7 days after PIN treatment, a significant partial inhibition of the erectile response was observed. Electroporation per se does not affect the erectile response to EFS at periods longer than 2–3 days [12]. However, administration of 30 µg of the ß form of PnNOS (deleted for exon 2; hence, the protein will not bind PIN) failed to induce correction of the erectile dysfunction induced in turn by the form in old rats (Fig. 10, right). This is despite the prediction that this treatment with the truncated PnNOS should be refractory to PIN and, therefore, that it should be more efficient than a construct expressing the exon 2 region that binds PIN.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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These results, demonstrating the coexistence of PIN and PnNOS in certain neurons in the corpora cavernosa, are in agreement with similar findings in the hypothalamus, specifically in the PVN (peniventricular nucleus) and MPOA (medial preoptic area) regions involved in the central control of erection, ejaculation, and reproductive behavior [20]. This suggests a possible role for PIN in both the central and peripheral control of nitrergic neurotransmission in the penile erectile response. This assumption is, however, tempered by the failure of recombinant PIN-GST to inhibit NOS activity or to bind to nNOS in penile extracts, which was expected from interaction studies with recombinant nNOS [16] in which PIN-GST and PIN were equivalent in their ability to inhibit NOS. Indeed, contradictory reports have been published regarding the ability of PIN to inhibit NO synthesis. The original study showed an IC50 of approximately 1 µM for GST-PIN with nNOS [16] and a supposedly specific effect on this isoform, whereas a subsequent study showed an IC50 that was 20-fold higher and no specificity as regards eNOS (endothelial nitric oxide synthese) and iNOS (inducible nitrix oxide synthase) [21]. A third study found that PIN does bind to nNOS but does not inhibit nNOS activity or dissociate the nNOS dimer into monomers [22]. It is possible that in incubations performed with tissue extracts, as in our case, other endogenous regulators may be present that would interfere with PIN/nNOS interaction, and this would not be detected by assays using recombinant nNOS. Another uncertainty may arise from the unresolved question of eNOS sensitivity to PIN [16, 21]. If eNOS is refractory to PIN [16], then the presence of eNOS in the penile homogenate would cause total NOS activity to be partially refractory to PIN, independent from factors that may interfere with PIN/PnNOS interaction.
On the other hand, it has been proposed that PIN, being a light-chain cytoplasmic dynein (DLC8), is involved in nNOS axonal transport [34, 35]. Dyneins are multi-subunit molecular motors that translocate molecular cargoes along microtubules [28, 29]. The PIN sequence is also shared by the nonmuscular myosin V associated with actin [36], so both actin- and microtubule-based molecular motors have a common protein component. It is possible, therefore, that PIN may participate in axonal transport in nerve fibers, but how this would affect NO synthesis is not clear. In addition, PIN binds to inhibitors of protein kinase A, an enzyme that specifically phosphorylates nNOS [37], thus suggesting that PIN may mediate nNOS phosphorylation and affect nNOS activity.
The partial inhibition of the erectile response exerted by PIN cDNA injected and electroporated 7 days before the EFS assay would support some role for PIN in inhibiting the nitrergic transmission for erection by reducing NO synthesis in vivo in the penis. The injection/electroporation approach has been validated in our previous study regarding gene therapy of erectile dysfunction in aged rats with plasmid and adenoviral constructs of the full-length form of PnNOS [12]. The homologous plasmid constructs for ß-galactosidase as a reporter gene are partially expressed in neural tissue in the penis [12], so it is reasonable to expect that the recombinant PIN may have been taken up and expressed by the nerve terminals, in which it should act as a modulator of PnNOS activity. However, construct expression also occurs in the cavernosal smooth muscle, in which the PIN/PnNOS interaction may not occur. In any case, inhibition of the erectile response by PIN in the present study was only moderate, and NOS activity in the penis of these rats could not be measured because of artifacts caused by EFS on this assay in penile homogenates [38]. Therefore, the interpretation that PIN affects PnNOS activity in vivo needs further investigation.
The fact that injection/electroporation of a plasmid construct of the truncated ß form of PnNOS to the corpora cavernosa of aged rats failed to correct the erectile response, contrary to the form, probably reflects a general lack of NOS activity of this isoform. The hypothesis for employing the ß form was based on its mRNA lacking exon 2 encoding the PDZ domain and, specifically, the amino acids 228–244 of nNOS that bind PIN [8]. Therefore, PnNOSß should be refractory to PIN action and more active in restoring erectile function compared to the form. However, the literature provides little evidence comparing the enzyme activity of both isoforms, so the question of whether the ß form is as active in vivo as the form remains open.
The association of an NMDAR subunit 2B in rat and mouse penile nerves with PnNOS gives more credence to the interpretation that the NMDAR subunits previously detected in this organ, including a truncated NMDAR1 mRNA [8], may be involved in a functional postsynaptic receptor that may participate in PnNOS activation through Ca2+ gating. However, the relaxing effects of NMDAR antagonists on penile strips in organ bath [8] would be contradictory to this concept; therefore, further work is needed to define the role of the NMDAR in the penis. This is particularly intriguing considering that NMDAR expression is restricted to the cavernosal nerve, is involved in eliciting centrally mediated erection, and is absent in the dorsal nerve, which is involved in reflexogenic erections [39].
In conclusion, the identification of PIN in PnNOS-expressing neurons in the pelvic ganglion and cavernosal and dorsal nerves and the effect of PIN cDNA on the erectile response to EFS suggest that their predicted protein-protein regulatory interaction may occur in the penile innervation and that this may affect nitrergic neurotransmission involved in penile erection. However, further studies are necessary to clarify whether this occurs by the postulated effect of PIN on nNOS dimerization and activity or through some other mechanism related to its participation in the cytoskeleton and its putative role in nNOS subcellular translocation and/or axonal transport. Similarly, the colocalization of NMDAR with PnNOS in the cavernosal nerve would allow us to assume a functional role for NMDAR similar to that in postsynaptic retrograde transmission in the central nervous system, but this remains to be established.
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FOOTNOTES |
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2 Correspondence: Nestor F. Gonzalez-Cadavid, Harbor-UCLA Research and Education Institute, Urology, Bldg. F-6, 1124 West Carson Street, Torrance, CA 90502. FAX: 310 222 1914; ncadavid{at}ucla.edu
Received: 13 May 2002.
First decision: 20 June 2002.
Accepted: 22 August 2002.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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  H. H. Davila, T. R. Magee, D. Vernet, J. Rajfer, and N. F. Gonzalez-Cadavid Gene Transfer of Inducible Nitric Oxide Synthase Complementary DNA Regresses the Fibrotic Plaque in an Animal Model of Peyronie's Disease Biol Reprod, November 1, 2004; 71(5): 1568 - 1577. [Abstract] [Full Text] [PDF]
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