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Lobe-specific Expression of Phosphodiesterase 5 in Rat Prostate
Basic and Translational Science Lobe-specific Expression of Phosphodiesterase 5 in Rat Prostate Lin Wang, Xiaoyu Zhang, Guifang Wang, Sandhya S. Visweswariah, Guiting Lin, Zhongcheng Xin, Tom F. Lue, and Ching-Shwun Lin OBJECTIVE METHODS
RESULTS
CONCLUSION
To investigate the level and location of phosphodiesterase 5 (PDE5) expression in rat prostate. The ventral, dorsal, and lateral lobes of rat prostate were examined for PDE5 expression by Western blotting. Intact rat urogenital complex, including the urinary bladder and accessory reproductive glands, was examined for PDE5 expression by immunohistochemistry. Individual prostatic lobes were further examined by immunofluorescence for expression of PDE5, a-smooth muscle actin, and rat endothelial cell antigen. Western blot analysis showed that PDE5 was expressed at a significantly lower level in dorsal lobe (DL) than in ventral lobe (VL) or lateral lobe (LL). Immunohistochemistry and immunofluorescence analyses showed that PDE5 was expressed in both acinar epithelium and periacinar smooth muscle. However, although similar levels of smooth muscle PDE5 expression were observed in all 3 prostatic lobes, significantly lower level of epithelial PDE5 expression was found in DL compared with VL or LL. In prostatic blood vessels, PDE5 expression was clearly visible in the endothelium but not as easily detectable in the smooth muscle. PDE5 was expressed in the acinar epithelium and periacinar smooth muscle of rat prostate. However, the epithelial PDE5 expression was significantly less in DL than in VL or LL. Regardless, the acinar wall, not the blood vessel wall, is the predominant PDE5 expression site in rat prostate. UROLOGY 85: 703.e7e703.e13, 2015. 2015 Elsevier Inc.
P
hosphodiesterase 5 (PDE5) is an enzyme that catalyzes the hydrolysis of cyclic guanosine monophosphate, and this activity is essential for smooth muscleecontaining organs, such as the penis, to transit from the relaxed to the contracted state.1 Inhibition of this activity with pharmacologic agents, known as PDE5 inhibitors (PDE5Is), has a high success rate of treating erectile dysfunction.2 PDE5 has also been identified in other lower urinary tract organs; thus, considerable interests have developed in the urology field to apply PDE5Is for the treatment of smooth muscleerelated dysfunctions of these organs.3-5 In 2011, one of such efforts resulted in the US Food and Drug Administration’s approval of tadalafil, a PDE5I, for the treatment of signs and symptoms of benign prostate hyperplasia. However, despite this approval, Lin Wang and Xiaoyu Zhang contributed equally to the study. Financial Disclosure: Sandhya S. Visweswariah received funding from the Council of Scientific and Industrial Research, Government of India. The other authors declare that they have no relevant financial interests. From the Knuppe Molecular Urology Laboratory, Department of Urology, University of California, San Francisco, CA; the Molecular Biology Laboratory, Andrology Center, Peking University First Hospital, Peking University, Beijing, China; the Department of Urology, Peking University First Hospital and the Institute of Urology, Peking University, Beijing, China; and the Department of Molecular Reproduction, Development and Genetics, Indian Institute of Science, Bangalore, India Address correspondence to: Ching-Shwun Lin, Ph.D., Department of Urology, University of California, San Francisco, CA 94143-0738. E-mail: [email protected]. edu Submitted: September 29, 2014, accepted (with revisions): December 3, 2014
ª 2015 Elsevier Inc. All Rights Reserved
questions have been raised about tadalafil’s mechanism of action because no statistical difference in maximum urinary flow rate was observed between tadalafil- and placebo-treated patients.6,7 Furthermore, published data concerning the level and location of PDE5 expression in the prostate have been highly inconsistent.5 In regard to PDE5 localization in human prostate, Uckert et al8 first reported that although a significant amount was detected in the fibromuscular stroma, the majority appeared to be associated with glandular structures. However, in a later study, Zenzmaier et al9 reported that PDE5 was predominantly expressed in the fibromuscular stroma, whereas no expression was detectable in the epithelium. In addition, 2 other independent studies by the same research group also failed to detect PDE5 expression in the epithelium.10,11 But, in this instance, PDE5 expression was determined to be scant in the fibromuscular stroma but predominant in vascular smooth muscle and endothelial cells. However, it should be noted that in these 2 studies the supporting histologic images were acquired respectively at 10 and 4 magnifications, which are too low for discerning the endothelium from the smooth muscle. In regard to PDE5 expression in rat prostate, Morelli et al11 again reported its presence in vascular endothelial and smooth muscle cells and its absence in the epithelium. However, again, the supporting histologic images were acquired at a low magnification of 4, and although http://dx.doi.org/10.1016/j.urology.2014.12.005 0090-4295/15
703.e7
the fibromuscular stroma was not mentioned, this particular tissue appears to have little or no stain. Regardless, in a later study, Zhang et al12 reported that PDE5 expression was scantly detected in the fibromuscular stroma. More significantly, although in this instance the histologic images were acquired at 100 and 400 magnifications, there was no mention of PDE5 expression in vascular endothelial or smooth muscle cells. As such, although these 2 studies agree on the absence of PDE5 expression in the epithelium, they disagree on whether PDE5 is expressed in the fibromuscular stroma or in blood vessels. In addition, it is also paradoxical that, even within the same study, PDE5 expression was identified as scant and abundant, respectively, by immunohistochemical (IHC) staining and Western blot analysis.12 In each of the 5 studies mentioned in the previous 2 paragraphs, prostatic PDE5 expression was investigated only as a companion or side issue. In fact, regardless of the findings, the location of PDE5 expression has little or no relevance to these studies’ main outcomes. However, when put together for reference or review, these studies cause considerable confusions. As such, we conducted the present study in hope of providing an accurate and comprehensive assessment of PDE5 expression in rat prostate. The resulting data indicate that PDE5 is expressed in a lobe-specific manner.
METHODS Antibodies Anti-PDE5 antibody is polyclonal rabbit antiserum generated against the first 138 amino acids of bovine PDE5A113 and has been used previously to detect PDE5 expression histologically in rat bladder, urethra, and penis.14,15 Anti-a smooth muscle actin (a-SMA), anti-rat endothelial cell antigen (RECA), and antib-actin antibodies were purchased from Abcam Inc. (Cambridge, MA), AbD Serotec (Raleigh, NC), and Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), respectively. Horseradish peroxidaseeconjugated goat antirabbit IgG antibodies was purchased from Thermo Fisher Scientific, Inc. (Rockford, IL). Alexa488-conjugated goat antirabbit IgG antibodies and Alexa594-conjugated goat antimouse IgG antibodies were purchased from Life Technologies Corp. (Carlsbad, CA).
Isolation of Rat Urogenital Complex All animal care and experimental procedures were approved by the institutional animal care and use committee at our institution. Six 14-week-old male Sprague-Dawley rats were obtained from Charles River Laboratories (Wilmington, MA) and identically processed. They were sacrificed by pentobarbital overdose, and their urogenital complexes were isolated according to Jesik et al.16 Three of the 6 isolated urogenital complexes were used for Western blot analysis, whereas the other 3 were used for immunohistochemical and immunofluorescence (IF) staining.
Western Blot Analysis Tissue samples were dissected from prostatic ventral, dorsal, and lateral lobes and homogenized in Radio-Immunoprecipitation Assay buffer. Equal amount (20 mg) from each sample was electrophoresed in 10% sodium dodecyl sulfate polyacrylamide 703.e8
gel electrophoresis. The fractionated proteins were then transferred to Polyvinylidene Difluoride membrane (Millipore Corp., Bedford, MA), which was then incubated with anti-PDE5 or anti-b-actin antibody overnight at 4 C, followed by incubation with horseradish peroxidaseeconjugated goat antirabbit IgG antibody for 1 hour at room temperature. Detection of reactive antigens was performed with an Enhanced Chemiluminescence kit (Amersham Life Sciences Inc., Arlington Heights, IL). The resulting image was analyzed with ChemiImager 4000 (Alpha Innotech, San Leandro, CA) to determine the integrated density value of each protein band.
IHC and IF Staining The urogenital complex was fixed for 4 hours in cold 2% formaldehyde and 0.002% saturated picric acid in 0.1-M phosphate buffer, pH 8.0, followed by overnight immersion in buffer containing 30% sucrose. The specimens were then embedded in optimal cutting temperature compound (Sakura Finetic USA, Torrance, CA) and stored at 80 C until use. Sections were cut at 5 m, mounted onto SuperFrost Plusecharged slides (Fisher Scientific, Pittsburgh, PA), and air dried for 5 minutes. For IHC staining, tissue sections were incubated in 3% hydrogen peroxide and methanol for 10 minutes to eliminate endogenous peroxidase activity. The sections were then incubated in 3% horse serum in phosphate-buffered saline (PBS) and 0.3% Triton X-100 for 30 minutes, followed by incubation with antiPDE5 antibody or rabbit serum (negative control) overnight. After rinses with PBS, the sections were stained with an avidinbiotin enzyme complex kit (VECTASTAIN, Vector Laboratories, Burlingame, CA) that uses diaminobenzidine as the chromogen (brown color) for indication of positive immunoreaction. The sections were then counterstained with hematoxylin. For IF staining, the tissue sections were incubated in 0.3% hydrogen peroxide and methanol for 10 minutes, washed twice in PBS for 5 minutes, and incubated with 3% horse serum in PBS or 0.3% Triton X-100 for 30 minutes at room temperature. After draining this solution from the tissue section, the slides were incubated at 4 C with anti-PDE5, anti-a-SMA, and/ or anti-RECA antibody overnight. Negative controls were similarly prepared except no primary antibody was added. After rinses with PBS, the sections were incubated with Alexa488conjugated goat antirabbit IgG antibody or Alexa594conjugated goat antimouse IgG antibody. After rinses with PBS, the slides were incubated with 1 mg/mL of 40 ,6-diamidino2-phenylindole (for nuclear staining, Sigma-Aldrich, St. Louis, MO).
Microscopy and Image Analysis The stained tissues were examined with a Nikon Eclipse E600 fluorescence microscope and photographed with a Retiga 1300 QImaging camera using the ACT-1 software (Nikon Instruments, Melville, NY). The photographs were then analyzed with Image-Pro Plus 6.0 software (Media Cybernetics, Silver Spring, MD). For quantification of PDE5 expression, five 400 microscopic fields were chosen for each prostatic lobe from tissues double-stained with anti-PDE5 and anti-a-SMA antibodies. Amount of PDE5 expression was determined by counting the pixel numbers of green stains on acinar walls. The counting was performed separately for the epithelium and the smooth muscle, which were defined respectively by the absence and presence of red stains on the same acinar wall. UROLOGY 85 (3), 2015
Figure 1. Lobe-specific differential expression of phosphodiesterase 5 (PDE5) in rat prostate. (A) Individual prostatic lobes were isolated from each of the 3 rats and analyzed by Western blotting for PDE5 expression, with b-actin serving as reference protein. (B) The protein bands were quantified by densitometry and expressed as PDE5-to-b-actin ratio for comparison among different prostatic lobes. (C) Sagittal sections of rat prostatic complex were stained with (left) and without (right) anti-PDE5 antibody and photographed at 20 magnification. Brown stains indicate positive PDE5 expression. B, urinary bladder; DL, dorsal lobe; LL, lateral lobe; SV, seminal vesicle; VL, ventral lobe.
Statistical Analysis Data were analyzed with Prism 5 (GraphPad Software, Inc., San Diego, CA) and shown as the mean standard error of the mean. Student unpaired t test was used to evaluate significance of difference, with P <.05 indicating significant difference.
RESULTS Differential PDE5 Expression in Different Prostatic Lobes For testing whether PDE5 is differentially expressed in different prostatic lobes, individual lobes were isolated from 3 rats and analyzed by Western blotting. The results showed that the dorsal lobe (DL) expressed PDE5 at a significantly lower level (P <.05) than ventral lobe (VL) or lateral lobe (LL), whereas no statistical difference (P >.05) was found between VL and LL (Fig. 1A,B). For further testing by IHC staining, the intact prostatic complex, including the urinary bladder, was isolated from each of 3 additional rats, sectioned sagittally through all 3 lobes, and stained for PDE5. At 20 magnification, UROLOGY 85 (3), 2015
positive staining was clearly visible on the acini in the LL and VL but not as certain in the DL (Fig. 1C). Differential PDE5 Expression in Epithelium and Smooth Muscle At higher magnifications, positive staining was seen on the entire thickness of the acinar wall in the VL and LL but only on the outer 1 quarter to one fifth of the acinar wall in the DL (Fig. 2). Further examination by IF costaining for PDE5 and a-SMA clearly showed that PDE5 was expressed on both the acinar epithelium and periacinar smooth muscle in the VL and LL (Fig. 3). On the other hand, PDE5 was mainly expressed in the periacinar smooth muscle in the DL (Fig. 3). Thus, although PDE5 expression in periacinar smooth muscle was similar in different lobes, PDE5 expression in acinar epithelium was significantly lower (P <.05) in the DL than in the VL and LL (Fig. 3). PDE5 Expression in Prostatic Blood Vessels Prostatic blood vessels appeared weakly stained for PDE5 when examined by IHC (pointed at with black 703.e9
Figure 2. Identification of phosphodiesterase 5 (PDE5) expression in different prostatic lobes by immunohistochemistry. PDE5-stained sections of rat prostatic complex were photographed at 100 (left) and 1000 (right) magnifications. Brown stains indicate positive PDE5 expression. Arrowheads point at blood vessels in left middle and lower panels. DL, dorsal lobe; LL, lateral lobe; VL, ventral lobe.
arrowheads in Fig. 2). Further examination by IF showed that vascular smooth muscle was weakly stained for PDE5, whereas vascular endothelium was more strongly stained (Fig. 4).
COMMENT As described in the introductory section, 5 studies have examined PDE5 expression in the prostate with sharply different outcomes. Although the exact cause for such inconsistencies is presently unknown, one possibility is that each of these studies examined only a small tissue sample, however it is well known that different prostatic zones or lobes differ histologically and pathologically.17-19 In addition, lobe-specific protein expression patterns have been observed by proteomic gel electrophoresis as early as in 1978 and 1985.20,21 And, more recently, lobe-specific distribution of individual proteins has also been demonstrated by immunostaining and Western blot analysis; for example, H-K-ATPase type 222 and TIMP-2.23 Thus, when conflicting protein data occurred, as in the case of 703.e10
prostatic PDE5 expression, the possibility of regional specific expression needs to be considered. In the present study, lobe-specific expression of PDE5 could be seen clearly in Western blot analysis, with the DL expressing at a much lower level compared to VL or LL. IHC staining of sagittal sections of the prostatic complex also showed such a differential expression pattern. But more importantly, higher magnifications of the stained tissue and a-SMA costaining further showed that the differential expression was associated with the epithelium not the smooth muscle, which was stained at equal levels in different prostatic lobes. The differentiation between DL and the other 2 prostatic lobes is surprising as the DL and LL are often considered a single structure—the dorsolateral lobe— because of their anatomic proximity as well as morphologic and pathologic similarities.18 The epithelial expression of PDE5 is also unusual as few studies have shown such expression by means of immunostaining.1 The first such demonstration was in rat renal tubules and pancreatic ducts.24 The second was in rat penile UROLOGY 85 (3), 2015
Figure 3. Identification of phosphodiesterase 5 (PDE5) expression in rat prostatic complex by immunofluorescence. Sagittal sections of rat prostatic complex were double-stained with anti-PDE5 and anti-a smooth muscle actin (SMA) antibodies and photographed at 400 magnification. Green and red stains indicate PDE5 and a-SMA, respectively. Blue stains by 40 ,6diamidino-2-phenylindole (DAPI) indicate cell nuclei. Quantification of PDE5 expression was performed by counting the number of pixels in green-stained areas on the acinar walls. Separation of acinar epithelium from periacinar smooth muscle was based on red-stained areas, which define smooth muscle. DL, dorsal lobe; LL, lateral lobe; VL, ventral lobe.
urethra25 although our previous study detected no PDE5 expression in female rat urethral epithelium.15 The third was in human prostate8 although 3 other studies disagreed9-11 and another study also showed no PDE5 expression in rat prostatic epithelium.12 However, the present study for the first time demonstrated epithelial PDE5 expression in rat prostate and further showed that such expression was lobe specific. Unlike previous studies, we verified such expression by costaining for a-SMA, which enabled the clear separation of the acinar epithelium from the periacinar smooth muscle. Despite the scarcity of studies demonstrating epithelial PDE5 expression by immunostaining, many have identified such expression by other means such as Western UROLOGY 85 (3), 2015
blotting and enzymatic activity.1 In the case of intestinal epithelial cells, PDE5 has been shown to regulate fluid secretion.26 Thus, it is not surprising that the initial finding of possible PDE5 expression in prostatic epithelium led to the proposal that such expression might be associated with glandular secretory function.8 In a more recent study, infertile patients treated with a single dose of 10-mg vardenafil, a PDE5I, every other day for 15 days were found to have significantly increased semen volume (3.4 0.1 mL; n ¼ 67) when compared to pretreatment baseline (2.9 0.1 mL) or to untreated patients (3.0 0.1 mL; n ¼ 65).27 However, whether the increased semen volume is because of direct inhibition of PDE5 in prostatic epithelium is presently unknown. 703.e11
Figure 4. Identification of PDE5 expression in prostatic blood vessels. Sagittal sections of rat prostatic complex were doublestained with anti-PDE5 and anti-a smooth muscle action (SMA) antibodies or with anti-PDE5 and antierat endothelial cell antigen (RECA) antibodies. The stained tissues were then examined with a fluorescence microscope and photographed at 400 magnification. In the resulting images, green stains indicate PDE5, blue stains indicate cell nuclei, and red stains indicate either a-SMA (left panels) or RECA (right panels). Note blood clots in vessel lumens were stained strongly PDE5positive (left panels), as platelets are known to express PDE5 at high levels.1,24,28
Fibbi et al10 and Morelli et al11 reported that prostatic PDE5 expression was predominantly seen in vascular smooth and endothelial cells. However, their histologic images were acquired at 4 or 10, which are too low for discerning such cells. Therefore, in the present study we examined prostatic tissues not only at much higher magnifications but also with specific staining for smooth muscle and endothelial cells. Although easily identifiable with anti-a-SMA antibody, vascular smooth muscle was weakly stained by anti-PDE5 antibody. On the other hand, vascular endothelium was easily recognized by both anti-RECA and anti-PDE5 antibodies. Thus, the present study unambiguously demonstrated endothelial expression of PDE5 in the prostate. However, compared to the acinar wall, the blood vessel wall cannot be the predominant site of PDE5 expression. The main limitation of our study is its lack of a mechanistic component. Thus, future studies should explore why PDE5 is differentially expressed in different prostatic lobes and how such differential expression 703.e12
relates to prostatic function. Another limitation of this study is the uncertain degree of relevance in using rat prostate as a model for discerning human prostate’s pathophysiology. In particular, human prostate lacks grossly visible lobular divisions; therefore, the lobespecific PDE5 expression identified in this study may not have a human equivalent. However, human prostate has been shown to consist of different anatomic zones that seem to correspond to rat prostatic lobes18; therefore, future studies should investigate whether PDE5 is differentially expressed in human prostatic zones and whether such differential expression is clinically relevant.
CONCLUSION We have obtained evidence that PDE5 is expressed in both the acinar epithelium and the periacinar smooth muscle of rat prostate. Although periacinar smooth muscle PDE5 expression is similarly abundant in DL, LL, and VL, acinar epithelial PDE5 expression is significantly UROLOGY 85 (3), 2015
less abundant in DL than in VL or LL. Furthermore, we have found that the prostate’s blood vessel walls express PDE5 at a lower level than the acinar walls do. References 1. Lin CS, Lin G, Xin ZC, et al. Expression, distribution and regulation of phosphodiesterase 5. Curr Pharm Des. 2006;12:3439-3457. 2. Dorsey P, Keel C, Klavens M, et al. Phosphodiesterase type 5 (PDE5) inhibitors for the treatment of erectile dysfunction. Expert Opin Pharmacother. 2010;11:1109-1122. 3. Liu L, Zheng S, Han P, et al. Phosphodiesterase-5 inhibitors for lower urinary tract symptoms secondary to benign prostatic hyperplasia: a systematic review and meta-analysis. Urology. 2011;77: 123-129. 4. Laydner HK, Oliveira P, Oliveira CR, et al. Phosphodiesterase 5 inhibitors for lower urinary tract symptoms secondary to benign prostatic hyperplasia: a systematic review. BJU Int. 2011;107:11041109. 5. Lin CS, Albersen M, Xin Z, et al. Phosphodiesterase-5 expression and function in the lower urinary tract: a critical review. Urology. 2013;81:480-487. 6. Gacci M, Corona G, Salvi M, et al. A systematic review and metaanalysis on the use of phosphodiesterase 5 inhibitors alone or in combination with alpha-blockers for lower urinary tract symptoms due to benign prostatic hyperplasia. Eur Urol. 2012;61:994-1003. 7. Giuliano F, Uckert S, Maggi M, et al. The mechanism of action of phosphodiesterase type 5 inhibitors in the treatment of lower urinary tract symptoms related to benign prostatic hyperplasia. Eur Urol. 2013;63:506-516. 8. Uckert S, Oelke M, Stief CG, et al. Immunohistochemical distribution of cAMP- and cGMP-phosphodiesterase (PDE) isoenzymes in the human prostate. Eur Urol. 2006;49:740-745. 9. Zenzmaier C, Sampson N, Pernkopf D, et al. Attenuated proliferation and trans-differentiation of prostatic stromal cells indicate suitability of phosphodiesterase type 5 inhibitors for prevention and treatment of benign prostatic hyperplasia. Endocrinology. 2010;151: 3975-3984. 10. Fibbi B, Morelli A, Vignozzi L, et al. Characterization of phosphodiesterase type 5 expression and functional activity in the human male lower urinary tract. J Sex Med. 2010;7:59-69. 11. Morelli A, Sarchielli E, Comeglio P, et al. Phosphodiesterase type 5 expression in human and rat lower urinary tract tissues and the effect of tadalafil on prostate gland oxygenation in spontaneously hypertensive rats. J Sex Med. 2011;8:2746-2760. 12. Zhang X, Zang N, Wei Y, et al. Testosterone regulates smooth muscle contractile pathways in the rat prostate: emphasis on PDE5 signaling. Am J Physiol Endocrinol Metab. 2012;302:E243-E253.
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13. Bakre MM, Sopory S, Visweswariah SS. Expression and regulation of the cGMP-binding, cGMP-specific phosphodiesterase (PDE5) in human colonic epithelial cells: role in the induction of cellular refractoriness to the heat-stable enterotoxin peptide. J Cell Biochem. 2000;77:159-167. 14. Lin G, Hayashi N, Carrion R, et al. Improving erectile function by silencing phosphodiesterase-5. J Urol. 2005;174:1142-1148. 15. Lin G, Huang YC, Wang G, et al. Prominent expression of phosphodiesterase 5 in striated muscle of the rat urethra and levator ani. J Urol. 2010;184:769-774. 16. Jesik CJ, Holland JM, Lee C. An anatomic and histologic study of the rat prostate. Prostate. 1982;3:81-97. 17. Sluczanowska-Glabowska S, Laszczynska M, Wylot M, et al. Morphological and immunohistochemical comparison of three rat prostate lobes (lateral, dorsal and ventral) in experimental hyperprolactinemia. Folia Histochem Cytobiol. 2010;48:447-454. 18. Lee CH, Akin-Olugbade O, Kirschenbaum A. Overview of prostate anatomy, histology, and pathology. Endocrinol Metab Clin North Am. 2011;40:565-575. 19. Fine SW, Reuter VE. Anatomy of the prostate revisited: implications for prostate biopsy and zonal origins of prostate cancer. Histopathology. 2012;60:142-152. 20. Lee C, Tsai Y, Harrison HH, et al. Proteins of the rat prostate: I. Preliminary characterization by two-dimensional electrophoresis. Prostate. 1985;7:171-182. 21. Resnick MI. Soluble proteins of the rat prostate: electrophoretic separation. Invest Urol. 1978;15:358-360. 22. Pestov NB, Korneenko TV, Adams G, et al. Nongastric H-KATPase in rodent prostate: lobe-specific expression and apical localization. Am J Physiol Cell Physiol. 2002;282:C907-C916. 23. Delella FK, Justulin LA Jr, Felisbino SL. Tissue inhibitor of metalloproteinase-2 (TIMP-2) location in the ventral, lateral, dorsal and anterior lobes of rat prostate by immunohistochemistry. Cell Biol Int. 2007;31:229-234. 24. Kotera J, Fujishige K, Omori K. Immunohistochemical localization of cGMP-binding cGMP-specific phosphodiesterase (PDE5) in rat tissues. J Histochem Cytochem. 2000;48:685-693. 25. Morelli A, Filippi S, Mancina R, et al. Androgens regulate phosphodiesterase type 5 expression and functional activity in corpora cavernosa. Endocrinology. 2004;145:2253-2263. 26. Sopory S, Kaur T, Visweswariah SS. The cGMP-binding, cGMPspecific phosphodiesterase (PDE5): intestinal cell expression, regulation and role in fluid secretion. Cell Signal. 2004;16:681-692. 27. Rago R, Salacone P, Caponecchia L, et al. Effect of vardenafil on semen parameters in infertile men: a pilot study evaluating shortterm treatment. J Endocrinol Invest. 2012;35:897-900. 28. Ito M, Nishikawa M, Fujioka M, et al. Characterization of the isoenzymes of cyclic nucleotide phosphodiesterase in human platelets and the effects of E4021. Cell Signal. 1996;8:575-581.
703.e13
RESULTS
CONCLUSION
To investigate the level and location of phosphodiesterase 5 (PDE5) expression in rat prostate. The ventral, dorsal, and lateral lobes of rat prostate were examined for PDE5 expression by Western blotting. Intact rat urogenital complex, including the urinary bladder and accessory reproductive glands, was examined for PDE5 expression by immunohistochemistry. Individual prostatic lobes were further examined by immunofluorescence for expression of PDE5, a-smooth muscle actin, and rat endothelial cell antigen. Western blot analysis showed that PDE5 was expressed at a significantly lower level in dorsal lobe (DL) than in ventral lobe (VL) or lateral lobe (LL). Immunohistochemistry and immunofluorescence analyses showed that PDE5 was expressed in both acinar epithelium and periacinar smooth muscle. However, although similar levels of smooth muscle PDE5 expression were observed in all 3 prostatic lobes, significantly lower level of epithelial PDE5 expression was found in DL compared with VL or LL. In prostatic blood vessels, PDE5 expression was clearly visible in the endothelium but not as easily detectable in the smooth muscle. PDE5 was expressed in the acinar epithelium and periacinar smooth muscle of rat prostate. However, the epithelial PDE5 expression was significantly less in DL than in VL or LL. Regardless, the acinar wall, not the blood vessel wall, is the predominant PDE5 expression site in rat prostate. UROLOGY 85: 703.e7e703.e13, 2015. 2015 Elsevier Inc.
P
hosphodiesterase 5 (PDE5) is an enzyme that catalyzes the hydrolysis of cyclic guanosine monophosphate, and this activity is essential for smooth muscleecontaining organs, such as the penis, to transit from the relaxed to the contracted state.1 Inhibition of this activity with pharmacologic agents, known as PDE5 inhibitors (PDE5Is), has a high success rate of treating erectile dysfunction.2 PDE5 has also been identified in other lower urinary tract organs; thus, considerable interests have developed in the urology field to apply PDE5Is for the treatment of smooth muscleerelated dysfunctions of these organs.3-5 In 2011, one of such efforts resulted in the US Food and Drug Administration’s approval of tadalafil, a PDE5I, for the treatment of signs and symptoms of benign prostate hyperplasia. However, despite this approval, Lin Wang and Xiaoyu Zhang contributed equally to the study. Financial Disclosure: Sandhya S. Visweswariah received funding from the Council of Scientific and Industrial Research, Government of India. The other authors declare that they have no relevant financial interests. From the Knuppe Molecular Urology Laboratory, Department of Urology, University of California, San Francisco, CA; the Molecular Biology Laboratory, Andrology Center, Peking University First Hospital, Peking University, Beijing, China; the Department of Urology, Peking University First Hospital and the Institute of Urology, Peking University, Beijing, China; and the Department of Molecular Reproduction, Development and Genetics, Indian Institute of Science, Bangalore, India Address correspondence to: Ching-Shwun Lin, Ph.D., Department of Urology, University of California, San Francisco, CA 94143-0738. E-mail: [email protected]. edu Submitted: September 29, 2014, accepted (with revisions): December 3, 2014
ª 2015 Elsevier Inc. All Rights Reserved
questions have been raised about tadalafil’s mechanism of action because no statistical difference in maximum urinary flow rate was observed between tadalafil- and placebo-treated patients.6,7 Furthermore, published data concerning the level and location of PDE5 expression in the prostate have been highly inconsistent.5 In regard to PDE5 localization in human prostate, Uckert et al8 first reported that although a significant amount was detected in the fibromuscular stroma, the majority appeared to be associated with glandular structures. However, in a later study, Zenzmaier et al9 reported that PDE5 was predominantly expressed in the fibromuscular stroma, whereas no expression was detectable in the epithelium. In addition, 2 other independent studies by the same research group also failed to detect PDE5 expression in the epithelium.10,11 But, in this instance, PDE5 expression was determined to be scant in the fibromuscular stroma but predominant in vascular smooth muscle and endothelial cells. However, it should be noted that in these 2 studies the supporting histologic images were acquired respectively at 10 and 4 magnifications, which are too low for discerning the endothelium from the smooth muscle. In regard to PDE5 expression in rat prostate, Morelli et al11 again reported its presence in vascular endothelial and smooth muscle cells and its absence in the epithelium. However, again, the supporting histologic images were acquired at a low magnification of 4, and although http://dx.doi.org/10.1016/j.urology.2014.12.005 0090-4295/15
703.e7
the fibromuscular stroma was not mentioned, this particular tissue appears to have little or no stain. Regardless, in a later study, Zhang et al12 reported that PDE5 expression was scantly detected in the fibromuscular stroma. More significantly, although in this instance the histologic images were acquired at 100 and 400 magnifications, there was no mention of PDE5 expression in vascular endothelial or smooth muscle cells. As such, although these 2 studies agree on the absence of PDE5 expression in the epithelium, they disagree on whether PDE5 is expressed in the fibromuscular stroma or in blood vessels. In addition, it is also paradoxical that, even within the same study, PDE5 expression was identified as scant and abundant, respectively, by immunohistochemical (IHC) staining and Western blot analysis.12 In each of the 5 studies mentioned in the previous 2 paragraphs, prostatic PDE5 expression was investigated only as a companion or side issue. In fact, regardless of the findings, the location of PDE5 expression has little or no relevance to these studies’ main outcomes. However, when put together for reference or review, these studies cause considerable confusions. As such, we conducted the present study in hope of providing an accurate and comprehensive assessment of PDE5 expression in rat prostate. The resulting data indicate that PDE5 is expressed in a lobe-specific manner.
METHODS Antibodies Anti-PDE5 antibody is polyclonal rabbit antiserum generated against the first 138 amino acids of bovine PDE5A113 and has been used previously to detect PDE5 expression histologically in rat bladder, urethra, and penis.14,15 Anti-a smooth muscle actin (a-SMA), anti-rat endothelial cell antigen (RECA), and antib-actin antibodies were purchased from Abcam Inc. (Cambridge, MA), AbD Serotec (Raleigh, NC), and Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), respectively. Horseradish peroxidaseeconjugated goat antirabbit IgG antibodies was purchased from Thermo Fisher Scientific, Inc. (Rockford, IL). Alexa488-conjugated goat antirabbit IgG antibodies and Alexa594-conjugated goat antimouse IgG antibodies were purchased from Life Technologies Corp. (Carlsbad, CA).
Isolation of Rat Urogenital Complex All animal care and experimental procedures were approved by the institutional animal care and use committee at our institution. Six 14-week-old male Sprague-Dawley rats were obtained from Charles River Laboratories (Wilmington, MA) and identically processed. They were sacrificed by pentobarbital overdose, and their urogenital complexes were isolated according to Jesik et al.16 Three of the 6 isolated urogenital complexes were used for Western blot analysis, whereas the other 3 were used for immunohistochemical and immunofluorescence (IF) staining.
Western Blot Analysis Tissue samples were dissected from prostatic ventral, dorsal, and lateral lobes and homogenized in Radio-Immunoprecipitation Assay buffer. Equal amount (20 mg) from each sample was electrophoresed in 10% sodium dodecyl sulfate polyacrylamide 703.e8
gel electrophoresis. The fractionated proteins were then transferred to Polyvinylidene Difluoride membrane (Millipore Corp., Bedford, MA), which was then incubated with anti-PDE5 or anti-b-actin antibody overnight at 4 C, followed by incubation with horseradish peroxidaseeconjugated goat antirabbit IgG antibody for 1 hour at room temperature. Detection of reactive antigens was performed with an Enhanced Chemiluminescence kit (Amersham Life Sciences Inc., Arlington Heights, IL). The resulting image was analyzed with ChemiImager 4000 (Alpha Innotech, San Leandro, CA) to determine the integrated density value of each protein band.
IHC and IF Staining The urogenital complex was fixed for 4 hours in cold 2% formaldehyde and 0.002% saturated picric acid in 0.1-M phosphate buffer, pH 8.0, followed by overnight immersion in buffer containing 30% sucrose. The specimens were then embedded in optimal cutting temperature compound (Sakura Finetic USA, Torrance, CA) and stored at 80 C until use. Sections were cut at 5 m, mounted onto SuperFrost Plusecharged slides (Fisher Scientific, Pittsburgh, PA), and air dried for 5 minutes. For IHC staining, tissue sections were incubated in 3% hydrogen peroxide and methanol for 10 minutes to eliminate endogenous peroxidase activity. The sections were then incubated in 3% horse serum in phosphate-buffered saline (PBS) and 0.3% Triton X-100 for 30 minutes, followed by incubation with antiPDE5 antibody or rabbit serum (negative control) overnight. After rinses with PBS, the sections were stained with an avidinbiotin enzyme complex kit (VECTASTAIN, Vector Laboratories, Burlingame, CA) that uses diaminobenzidine as the chromogen (brown color) for indication of positive immunoreaction. The sections were then counterstained with hematoxylin. For IF staining, the tissue sections were incubated in 0.3% hydrogen peroxide and methanol for 10 minutes, washed twice in PBS for 5 minutes, and incubated with 3% horse serum in PBS or 0.3% Triton X-100 for 30 minutes at room temperature. After draining this solution from the tissue section, the slides were incubated at 4 C with anti-PDE5, anti-a-SMA, and/ or anti-RECA antibody overnight. Negative controls were similarly prepared except no primary antibody was added. After rinses with PBS, the sections were incubated with Alexa488conjugated goat antirabbit IgG antibody or Alexa594conjugated goat antimouse IgG antibody. After rinses with PBS, the slides were incubated with 1 mg/mL of 40 ,6-diamidino2-phenylindole (for nuclear staining, Sigma-Aldrich, St. Louis, MO).
Microscopy and Image Analysis The stained tissues were examined with a Nikon Eclipse E600 fluorescence microscope and photographed with a Retiga 1300 QImaging camera using the ACT-1 software (Nikon Instruments, Melville, NY). The photographs were then analyzed with Image-Pro Plus 6.0 software (Media Cybernetics, Silver Spring, MD). For quantification of PDE5 expression, five 400 microscopic fields were chosen for each prostatic lobe from tissues double-stained with anti-PDE5 and anti-a-SMA antibodies. Amount of PDE5 expression was determined by counting the pixel numbers of green stains on acinar walls. The counting was performed separately for the epithelium and the smooth muscle, which were defined respectively by the absence and presence of red stains on the same acinar wall. UROLOGY 85 (3), 2015
Figure 1. Lobe-specific differential expression of phosphodiesterase 5 (PDE5) in rat prostate. (A) Individual prostatic lobes were isolated from each of the 3 rats and analyzed by Western blotting for PDE5 expression, with b-actin serving as reference protein. (B) The protein bands were quantified by densitometry and expressed as PDE5-to-b-actin ratio for comparison among different prostatic lobes. (C) Sagittal sections of rat prostatic complex were stained with (left) and without (right) anti-PDE5 antibody and photographed at 20 magnification. Brown stains indicate positive PDE5 expression. B, urinary bladder; DL, dorsal lobe; LL, lateral lobe; SV, seminal vesicle; VL, ventral lobe.
Statistical Analysis Data were analyzed with Prism 5 (GraphPad Software, Inc., San Diego, CA) and shown as the mean standard error of the mean. Student unpaired t test was used to evaluate significance of difference, with P <.05 indicating significant difference.
RESULTS Differential PDE5 Expression in Different Prostatic Lobes For testing whether PDE5 is differentially expressed in different prostatic lobes, individual lobes were isolated from 3 rats and analyzed by Western blotting. The results showed that the dorsal lobe (DL) expressed PDE5 at a significantly lower level (P <.05) than ventral lobe (VL) or lateral lobe (LL), whereas no statistical difference (P >.05) was found between VL and LL (Fig. 1A,B). For further testing by IHC staining, the intact prostatic complex, including the urinary bladder, was isolated from each of 3 additional rats, sectioned sagittally through all 3 lobes, and stained for PDE5. At 20 magnification, UROLOGY 85 (3), 2015
positive staining was clearly visible on the acini in the LL and VL but not as certain in the DL (Fig. 1C). Differential PDE5 Expression in Epithelium and Smooth Muscle At higher magnifications, positive staining was seen on the entire thickness of the acinar wall in the VL and LL but only on the outer 1 quarter to one fifth of the acinar wall in the DL (Fig. 2). Further examination by IF costaining for PDE5 and a-SMA clearly showed that PDE5 was expressed on both the acinar epithelium and periacinar smooth muscle in the VL and LL (Fig. 3). On the other hand, PDE5 was mainly expressed in the periacinar smooth muscle in the DL (Fig. 3). Thus, although PDE5 expression in periacinar smooth muscle was similar in different lobes, PDE5 expression in acinar epithelium was significantly lower (P <.05) in the DL than in the VL and LL (Fig. 3). PDE5 Expression in Prostatic Blood Vessels Prostatic blood vessels appeared weakly stained for PDE5 when examined by IHC (pointed at with black 703.e9
Figure 2. Identification of phosphodiesterase 5 (PDE5) expression in different prostatic lobes by immunohistochemistry. PDE5-stained sections of rat prostatic complex were photographed at 100 (left) and 1000 (right) magnifications. Brown stains indicate positive PDE5 expression. Arrowheads point at blood vessels in left middle and lower panels. DL, dorsal lobe; LL, lateral lobe; VL, ventral lobe.
arrowheads in Fig. 2). Further examination by IF showed that vascular smooth muscle was weakly stained for PDE5, whereas vascular endothelium was more strongly stained (Fig. 4).
COMMENT As described in the introductory section, 5 studies have examined PDE5 expression in the prostate with sharply different outcomes. Although the exact cause for such inconsistencies is presently unknown, one possibility is that each of these studies examined only a small tissue sample, however it is well known that different prostatic zones or lobes differ histologically and pathologically.17-19 In addition, lobe-specific protein expression patterns have been observed by proteomic gel electrophoresis as early as in 1978 and 1985.20,21 And, more recently, lobe-specific distribution of individual proteins has also been demonstrated by immunostaining and Western blot analysis; for example, H-K-ATPase type 222 and TIMP-2.23 Thus, when conflicting protein data occurred, as in the case of 703.e10
prostatic PDE5 expression, the possibility of regional specific expression needs to be considered. In the present study, lobe-specific expression of PDE5 could be seen clearly in Western blot analysis, with the DL expressing at a much lower level compared to VL or LL. IHC staining of sagittal sections of the prostatic complex also showed such a differential expression pattern. But more importantly, higher magnifications of the stained tissue and a-SMA costaining further showed that the differential expression was associated with the epithelium not the smooth muscle, which was stained at equal levels in different prostatic lobes. The differentiation between DL and the other 2 prostatic lobes is surprising as the DL and LL are often considered a single structure—the dorsolateral lobe— because of their anatomic proximity as well as morphologic and pathologic similarities.18 The epithelial expression of PDE5 is also unusual as few studies have shown such expression by means of immunostaining.1 The first such demonstration was in rat renal tubules and pancreatic ducts.24 The second was in rat penile UROLOGY 85 (3), 2015
Figure 3. Identification of phosphodiesterase 5 (PDE5) expression in rat prostatic complex by immunofluorescence. Sagittal sections of rat prostatic complex were double-stained with anti-PDE5 and anti-a smooth muscle actin (SMA) antibodies and photographed at 400 magnification. Green and red stains indicate PDE5 and a-SMA, respectively. Blue stains by 40 ,6diamidino-2-phenylindole (DAPI) indicate cell nuclei. Quantification of PDE5 expression was performed by counting the number of pixels in green-stained areas on the acinar walls. Separation of acinar epithelium from periacinar smooth muscle was based on red-stained areas, which define smooth muscle. DL, dorsal lobe; LL, lateral lobe; VL, ventral lobe.
urethra25 although our previous study detected no PDE5 expression in female rat urethral epithelium.15 The third was in human prostate8 although 3 other studies disagreed9-11 and another study also showed no PDE5 expression in rat prostatic epithelium.12 However, the present study for the first time demonstrated epithelial PDE5 expression in rat prostate and further showed that such expression was lobe specific. Unlike previous studies, we verified such expression by costaining for a-SMA, which enabled the clear separation of the acinar epithelium from the periacinar smooth muscle. Despite the scarcity of studies demonstrating epithelial PDE5 expression by immunostaining, many have identified such expression by other means such as Western UROLOGY 85 (3), 2015
blotting and enzymatic activity.1 In the case of intestinal epithelial cells, PDE5 has been shown to regulate fluid secretion.26 Thus, it is not surprising that the initial finding of possible PDE5 expression in prostatic epithelium led to the proposal that such expression might be associated with glandular secretory function.8 In a more recent study, infertile patients treated with a single dose of 10-mg vardenafil, a PDE5I, every other day for 15 days were found to have significantly increased semen volume (3.4 0.1 mL; n ¼ 67) when compared to pretreatment baseline (2.9 0.1 mL) or to untreated patients (3.0 0.1 mL; n ¼ 65).27 However, whether the increased semen volume is because of direct inhibition of PDE5 in prostatic epithelium is presently unknown. 703.e11
Figure 4. Identification of PDE5 expression in prostatic blood vessels. Sagittal sections of rat prostatic complex were doublestained with anti-PDE5 and anti-a smooth muscle action (SMA) antibodies or with anti-PDE5 and antierat endothelial cell antigen (RECA) antibodies. The stained tissues were then examined with a fluorescence microscope and photographed at 400 magnification. In the resulting images, green stains indicate PDE5, blue stains indicate cell nuclei, and red stains indicate either a-SMA (left panels) or RECA (right panels). Note blood clots in vessel lumens were stained strongly PDE5positive (left panels), as platelets are known to express PDE5 at high levels.1,24,28
Fibbi et al10 and Morelli et al11 reported that prostatic PDE5 expression was predominantly seen in vascular smooth and endothelial cells. However, their histologic images were acquired at 4 or 10, which are too low for discerning such cells. Therefore, in the present study we examined prostatic tissues not only at much higher magnifications but also with specific staining for smooth muscle and endothelial cells. Although easily identifiable with anti-a-SMA antibody, vascular smooth muscle was weakly stained by anti-PDE5 antibody. On the other hand, vascular endothelium was easily recognized by both anti-RECA and anti-PDE5 antibodies. Thus, the present study unambiguously demonstrated endothelial expression of PDE5 in the prostate. However, compared to the acinar wall, the blood vessel wall cannot be the predominant site of PDE5 expression. The main limitation of our study is its lack of a mechanistic component. Thus, future studies should explore why PDE5 is differentially expressed in different prostatic lobes and how such differential expression 703.e12
relates to prostatic function. Another limitation of this study is the uncertain degree of relevance in using rat prostate as a model for discerning human prostate’s pathophysiology. In particular, human prostate lacks grossly visible lobular divisions; therefore, the lobespecific PDE5 expression identified in this study may not have a human equivalent. However, human prostate has been shown to consist of different anatomic zones that seem to correspond to rat prostatic lobes18; therefore, future studies should investigate whether PDE5 is differentially expressed in human prostatic zones and whether such differential expression is clinically relevant.
CONCLUSION We have obtained evidence that PDE5 is expressed in both the acinar epithelium and the periacinar smooth muscle of rat prostate. Although periacinar smooth muscle PDE5 expression is similarly abundant in DL, LL, and VL, acinar epithelial PDE5 expression is significantly UROLOGY 85 (3), 2015
less abundant in DL than in VL or LL. Furthermore, we have found that the prostate’s blood vessel walls express PDE5 at a lower level than the acinar walls do. References 1. Lin CS, Lin G, Xin ZC, et al. Expression, distribution and regulation of phosphodiesterase 5. Curr Pharm Des. 2006;12:3439-3457. 2. Dorsey P, Keel C, Klavens M, et al. Phosphodiesterase type 5 (PDE5) inhibitors for the treatment of erectile dysfunction. Expert Opin Pharmacother. 2010;11:1109-1122. 3. Liu L, Zheng S, Han P, et al. Phosphodiesterase-5 inhibitors for lower urinary tract symptoms secondary to benign prostatic hyperplasia: a systematic review and meta-analysis. Urology. 2011;77: 123-129. 4. Laydner HK, Oliveira P, Oliveira CR, et al. Phosphodiesterase 5 inhibitors for lower urinary tract symptoms secondary to benign prostatic hyperplasia: a systematic review. BJU Int. 2011;107:11041109. 5. Lin CS, Albersen M, Xin Z, et al. Phosphodiesterase-5 expression and function in the lower urinary tract: a critical review. Urology. 2013;81:480-487. 6. Gacci M, Corona G, Salvi M, et al. A systematic review and metaanalysis on the use of phosphodiesterase 5 inhibitors alone or in combination with alpha-blockers for lower urinary tract symptoms due to benign prostatic hyperplasia. Eur Urol. 2012;61:994-1003. 7. Giuliano F, Uckert S, Maggi M, et al. The mechanism of action of phosphodiesterase type 5 inhibitors in the treatment of lower urinary tract symptoms related to benign prostatic hyperplasia. Eur Urol. 2013;63:506-516. 8. Uckert S, Oelke M, Stief CG, et al. Immunohistochemical distribution of cAMP- and cGMP-phosphodiesterase (PDE) isoenzymes in the human prostate. Eur Urol. 2006;49:740-745. 9. Zenzmaier C, Sampson N, Pernkopf D, et al. Attenuated proliferation and trans-differentiation of prostatic stromal cells indicate suitability of phosphodiesterase type 5 inhibitors for prevention and treatment of benign prostatic hyperplasia. Endocrinology. 2010;151: 3975-3984. 10. Fibbi B, Morelli A, Vignozzi L, et al. Characterization of phosphodiesterase type 5 expression and functional activity in the human male lower urinary tract. J Sex Med. 2010;7:59-69. 11. Morelli A, Sarchielli E, Comeglio P, et al. Phosphodiesterase type 5 expression in human and rat lower urinary tract tissues and the effect of tadalafil on prostate gland oxygenation in spontaneously hypertensive rats. J Sex Med. 2011;8:2746-2760. 12. Zhang X, Zang N, Wei Y, et al. Testosterone regulates smooth muscle contractile pathways in the rat prostate: emphasis on PDE5 signaling. Am J Physiol Endocrinol Metab. 2012;302:E243-E253.
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