Synthesis and Secretion of Angiotensin II by the Prostate Gland in Vitro
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内分泌学杂志 2005年第1期
School of Biological Sciences, Queen Mary, University of London, London E1 4NS, United Kingdom
Address all correspondence and requests for reprints to: Dr. Gavin Vinson, School of Biological Sciences, Queen Mary, University of London, Mile End Road, London E1 4NS, United Kingdom. E-mail: g.p.vinson@qmul.ac.uk.
Abstract
The renin angiotensin system has been shown to have tissue-related functions that are distinct from its systemic roles. We showed that angiotensin II type 1 (AT1) receptors are present in mammalian sperm, and angiotensin II stimulates sperm motility and capacitation. In addition, angiotensin II is present in human seminal plasma at concentrations higher than found in blood. In testing the possibility that the prostate may be the source of seminal plasma angiotensin II, mRNA coding for angiotensinogen, (pro)renin, and angiotensin-converting enzyme were identified by RT-PCR in rat and human prostate and in prostate LNCaP cells, as well as the angiotensin receptors types 1 and 2 (AT1 and AT2) in human tissues and AT1 in rat. In human tissue, immunocytochemistry showed cellular colocalization of renin with the AT1 receptor in secretory epithelial cells. Confirmation of the capacity of the prostate to secrete angiotensin II was shown by the detection of immunoreactive angiotensin in media removed from rat prostate organ cultures and LNCaP cells. Rat prostate angiotensin secretion was enhanced by dihydrotestosterone, but LNCaP angiotensin was stimulated by estradiol. This stimulation was blocked by tamoxifen. Rat prostate AT1 receptor expression was much greater in prepuberal than in postpuberal rats but was not affected by a low-sodium diet. It was, however, significantly enhanced by captopril pretreatment. These findings all suggest the independence of prostate and systemic renin angiotensin system regulation. The data presented here suggest that the prostate may be a source of the secreted angiotensin II found in seminal plasma.
Introduction
IN RECENT YEARS, IT has become evident that the renin angiotensin system (RAS) has a far wider significance than suggested by its traditional roles in water and electrolyte homeostasis. In particular, many tissues have been shown to contain all of the elements of the RAS and can seemingly synthesize the active peptides, particularly angiotensin II, but sometimes including angiotensin III and IV, for purely paracrine activity. These tissues include adrenal, kidney, brain, and the cardiovascular systems (1, 2, 3, 4, 5, 6, 7).
In the reproductive tract, too, there is now considerable evidence that complete function depends on a locally functional RAS. In part, this has been shown by relative infertility in animals with disrupted genes coding for RAS components, such as angiotensin-converting enzyme (ACE) or angiotensinogen (8, 9, 10), but, in addition, the distribution of RAS components, receptors, and associated angiotensin activities demonstrate functions that are exclusively concerned with the reproductive process (11, 12, 13, 14).
In the male, the existence of a specific testis form of ACE has been known for some years (15, 16, 17), whereas RAS components have been described in various tissues, including testis, epididymis, coagulating gland, and prostate (18, 19, 20, 21). Both ACE and prorenin are found in seminal plasma (22, 23, 24, 25). In addition, the presence of angiotensin II type 1 (AT1) receptors in ejaculated rat and human sperm strongly suggested the possibility that angiotensin affects sperm function, and we reported the stimulation by angiotensin II of patterns of motility associated with capacitation in sperm from patients attending a fertility clinic. This action was mediated through the AT1 receptor (26, 27). In vitro, using the hamster oocyte penetration test, angiotensin II has also been shown to increase the oocyte-penetrating ability of both mouse and human sperm (28, 29). Importantly, we have shown that human seminal plasma contains angiotensin II in concentrations higher than in circulating blood plasma (30). Consequently it is appropriate to determine the source of this secreted angiotensin II.
Materials and Methods
Materials
Total human prostate RNA was purchased from Clontech Laboratories Inc. (Palo Alto, CA). LNCaP cells were purchased from the European Collection of Cell Cultures (Salisbury, UK). General laboratory chemicals and reagents were supplied from Sigma-Aldrich (Poole, Dorset UK) unless otherwise indicated. Archival paraffin sections of human trans-urethral prostate resections were obtained from St. Bartholomew’s and the Royal London Hospital Trust with ethical committee approval.
The monoclonal antibody 6313/G2, specific for the AT1 receptor, was generated in our laboratory. This antibody was raised to a synthetic peptide representing residues 8–17 of the N-terminal extracellular domain (31). A second AT1 antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). As described by the manufacturers, this polyclonal antibody, AT1 (N-10), was also raised against a peptide that maps near the amino-terminal of the AT1 receptor. The antihuman (pro)renin antibody (F37.2D12) was a gift from P. Corvol (Institut National de la Santé et de la Recherche Médicale, Paris, France) (32). The rat renin antibody (K 026E V), which was raised against pure mouse submandibular renin, was a gift from Prof. K. Poulsen (Institute for Anatomy and Physiology, Bulowsvej, Denmark). All secondary antibodies were purchased from the Dako Corp. (Carpinteria, CA).
Animal treatments
Male Wistar rats were obtained from commercial sources and maintained at Queen Mary, University of London according to Home Office guidelines. To investigate the regulation of AT1 receptor expression, animal treatments included dietary manipulation and ACE inhibition. Male Wistar rats, 12–14 wk old, were maintained on a diet of whole-meal flour supplemented with 1% Ca2CO3 and 1% NaCl for 2 wk. Low-sodium diets omitted the 1% NaCl. Such dietary sodium regulation of the RAS is well documented and has been used extensively to investigate both paracrine and endocrine regulation of RAS (33, 34). In a second study, captopril (0.5 mg/ml) was added to drinking water for a period of 1 wk. To investigate the potential influence of puberty on the RAS, prostates were removed from six 30-d-old animals (prepuberal) and were compared with those excised from 60-d-old (postpuberal) animals.
Cell culture
Human prostate LNCaP cells were maintained and propagated in RPMI 1640 medium, supplemented with 10% fetal bovine serum, 2 mM glutamine, 1 mM sodium pyruvate, 10 mM nonessential amino acids, 100 U/ml penicillin, and 100 mg/ml streptomycin sulfate.
Rat organ culture
Organ cultures of rat ventral prostate were prepared according to the method of Trowell (35), with some modifications. Rats were killed by stunning and cervical dislocation. Prostates were excised and immediately placed into Krebs-Ringer-HEPES-bicarbonate glucose buffer [130 mM NaCl, 3.2 mM KCl, 1.2 mM CaCl2·H2O, 0.5 mM MgSO4·7H20, 10 mM HEPES, 2 mM NaHCO3, 0.5 mM KH2PO4, and 12 mM glucose (pH 7.4)] with added antibiotics (125 U/ml penicillin, 0.125 mg/ml streptomycin, and 0.05 mg/ml gentomycin) on ice. Once in a sterile class II hood, the prostates were cut into pieces of approximately 1 mm3. Five to seven explants were placed in transwell-clear cell culture inserts (0.4-μm pore size, VWR, Lutterworth, Leicester, UK) and 1 ml phenol red-free medium 199 supplemented with 10% charcoal-stripped fetal bovine serum, penicillin (100 IU/ml), streptomycin sulfate (100 μg/ml), and glutamine (100 μg/ml) was added to each well. All samples were incubated in a humidified atmosphere of 95% O2/5% CO2 for a period of 24 h after which fresh medium was added. Twenty-four hours later, media samples were transferred to tubes containing a mixture of proteinase inhibitors [100 μl, 25 mM phenanthrolene, 125 mM Na2EDTA, 2 g/liter neomycin in 2% (vol/vol) ethanol, 5 mM bestatin, and 2.5 mM chymostatin] (36) and stored at –20 C until required for angiotensin II measurement. Explants were collected into 300 μl radioimmunoprecipitation assay (RIPA) buffer (1x PBS, 0.1% Nonidet P-40, 0.5% sodium deoxycholate, and 0.1% sodium dodecyl sulfate, containing 1 mg/ml phenylmethylsulfonylfluoride, 1 μg/ml aprotinin, and 1 μg/ml soybean trypsin inhibitor), homogenized (3 x 30 sec), and allowed to stand on ice for 30 min. Cellular debris was removed by centrifugation at 10,000 rpm for 10 min at 4 C. Protein content was assayed using the DC method (Bio-Rad Laboratories, Muenchen Germany).
Cell culture for angiotensin II assay
LNCaP cells were seeded into tissue culture flasks (T150 cm2) and, once confluent, serum-free RPMI 1640 medium was added for a period of 48 h. Cells were then stimulated for 24 h with dihydrotestosterone (DHT) (10–8 M) or estradiol (E2) (10–8 M) in the presence of tamoxifen (10–6 M). Media were removed and collected into the previously described inhibitor mix (1 ml) on ice to prevent degradation of angiotensin II. Cells were pelleted by centrifugation at 1000 x g for 5 min, and protein was assayed as above.
Angiotensin II RIA
Media collected from rat prostate cultures and the LNCaP cell line were processed using solid-phase extraction on ENVI-18 columns (Supelco, Poole, UK) as described previously (30) and eluants were lyophilized. Immunoreactive angiotensin II levels were then assayed using an RIA kit supplied by Bachem (UK) Ltd. (St. Helens, Merseyside, UK)
Immunocytochemistry
Archival human prostate sections (n = 12) were obtained from the Department of Morbid Anatomy (The Royal London Hospital, London, UK). Tissues were removed at the time of operation with appropriate consent and placed in 10% formalin for 24 h before routine automated paraffin wax embedding. Sections (8 μm) were mounted on slides, dewaxed in xylene, and rehydrated through graded alcohols. Endogenous peroxidase activity was blocked by 30-min incubation in 3% hydrogen peroxide in methanol. Antigen retrieval was performed by microwave treatment for 10 min in 0.5 M trisodium citrate (pH 6.0). Nonspecific staining was blocked using 0.25% casein in PBS (Dako protein block serum free; Dako Corp.) for 30 min at room temperature followed by incubation with antihuman AT1 receptor antibody (AT1 N-10; Santa Cruz Biotechnology), diluted 1:1000 in PBS supplemented with 5% normal goat serum), the antihuman (pro)renin antibody (dilution 1:750 in PBS) (31), or the AT1 receptor antibody 6313/G2 (1:1000 in PBS) (32) overnight at 4 C. Sections were washed in PBS and subsequently incubated with appropriate biotinylated secondary antibodies (1:200 in PBS) for 30 min at room temperature. Visualization of the antigens was achieved using the peroxidase streptABC complex and incubation with the peroxidase substrate 3',3'-diaminobenzidine. Specificity of the AT1 receptor antibodies was checked by preabsorption of the primary antibody with the appropriate peptide, and, for the (pro)renin antibody, the primary antibody was omitted.
To investigate immunolocalization of the AT1 receptor and renin in rat prostate, tissue was isolated from prepuberal and postpuberal male Wistar rats and fixed in molecular biology fixative (Streck Laboratories. Inc., Omaha, NE) for 16 h at room temperature. Tissues were dehydrated through an alcohol series and embedded in paraffin wax using an automatic tissue processor (Shandon Elliot Scientific Company Ltd., London, UK). Tissue sections were mounted on 3'-aminopropyltriethoxy-silane-coated slides and dried at 37 C overnight.
To prevent endogenous peroxidase activity and to check for nonspecific staining, rat prostate tissues were treated as for human prostate sections. Antigen retrieval was also performed by microwave treatment for 10 min in 0.5 M trisodium citrate (pH 6.0). To identify the AT1 receptor in rat prostate tissue, the same protocols and AT1-specific antibodies were used as for human prostate sections because both the polyclonal AT1 (N-10) and the monoclonal 6313/G2 AT1-specific antibody cross-react with human and rat AT1 receptors. To identify renin in the rat prostate, the rabbit antibody (K 026E V) was used at a 1:500 dilution in PBS at 4 C overnight. Secondary antibody incubation and antigen visualization was as used for human prostate samples.
Immunoblotting
LNCaP cells were grown to confluence, serum starved for 24 h, then stimulated with angiotensin II (10–10 to 10–6 M) for an additional 24 h. Cells were collected into 200 μl RIPA buffer, and total protein was prepared. Rat prostate tissues were collected and homogenized in RIPA buffer on ice. Total protein was prepared, and protein samples were separated on 10% sodium dodecyl sulfate-PAGE gels, incubated for 1.5 h at room temperature in 5% nonfat milk powder in Tris-buffered saline (with 1% Tween), and immunoblotted with AT1 receptor antibody (Santa Cruz Biotechnology) diluted 1:1000 and incubated overnight at 4 C. Immunoreactive bands were detected by enhanced chemiluminescence (Amersham, Hertsfordshire, UK) and exposure to film.
RT-PCR
Total RNA was extracted from confluent LNCaP cells and rat prostate homogenates using the RNeasy minikit (Qiagen Ltd., West Sussex, UK). Human and cell line RNA samples were quantified, and 5 μg of total RNA was added to a mixture of 1 μl of oligo(dT)15 (0.5 mg/ml) with an appropriate volume of ribonuclease-free water and heated to 70 C for 10 min followed by a quick chill on ice. To each tube, 1 μl of dNTP mix (dATP, dTTP, dGTP, and dCTP, each to a final concentration of 10 mM), 2 μl 0.1 M dithiothreitol, and 4 μl 5x first-strand buffer was added and heated to 42 C for a further 2 min. Reverse transcriptase Moloney murine leukemia virus-reverse transcriptase was then added, and each sample was incubated at 42 C for 50 min followed by 70 C for 15 min. PCR was carried out using the primers described in Table 1 at a final concentration of 1 pmol/μl.
TABLE 1. Details of human primers utilized in this study
For investigation of rat tissue samples, the Access RT-PCR system (Promega, Southampton, UK) was used. RT-PCR profiles are outlined in Table 2. PCR cycle number and the RNA concentration were optimized so that linearity was achieved with increasing cycle number and RNA concentration. Optimal conditions are described in Tables 1 and 2. PCR products were separated on 1.5% agarose gels and visualized under UV light.
Statistical analysis
All results are expressed as mean values ± SEM. Differences between control and experimental groups were analyzed using Student’s t test. Statistical significance was defined as P < 0.05.
Results
To confirm the possibility that the prostate might be a potential site for angiotensin II synthesis, evidence for the main RAS components was sought in rat and human prostate tissue and in human prostate cells. Results obtained by RT-PCR are illustrated in Fig. 1. These show that mRNAs for angiotensinogen, renin, ACE, and the AT1 receptor are all transcribed in rat prostate (Fig. 1A), the human prostate (Fig. 1B), and the human prostate epithelial LNCaP cells (Fig. 1C). AT2 mRNA was also identified in all human samples examined. The data are qualitative and not representative of the magnitude of expression of particular components because it was not normalized to a control primer set.
FIG. 1. RT-PCR demonstrated mRNA for all the RAS components in rat (A) and human prostate (B) and LNCaP cells (C). Single bands were obtained throughout (+). Negative control lanes (–) omitted reverse transcriptase. AGT, Angiotensinogen; Ren, renin; AT1 and AT2, AII type 1 and type 2 receptors.
In both the rat prostate (n = 6) (Fig. 2) and the human prostate (n = 12) (Fig. 3), the major site of AT1 receptor immunoactivity was the secretory epithelial cells lining the ducts, with faint reactions in parts of the stroma in human tissue. In human prostate samples, staining intensity did vary among patients, but staining was always observed in both epithelium and the stroma. Confirmation of the epithelial localization of the AT1 receptor was obtained by using a second AT1 receptor antibody as outlined in Materials and Methods. Further evidence for epithelial localization of AT1 receptor was provided by immunoblotting of the LNCaP prostate epithelial cell line protein with an AT1-specific antibody (Santa Cruz Biotechnology), which gave a single band at the appropriate size (data not shown). Evidence for the presence of the AT1 receptor in rat prostate was also obtained by immunoblotting (Fig. 4). Immunostaining of the human prostate using the (pro)renin antibody illustrated that (pro)renin colocalizes with the AT1 receptor in secretory epithelial cells (Fig. 3).
FIG. 2. Immunocytochemistry of 30- and 60- d rat prostate sections using the AT1 (N-10) Santa Cruz polyclonal antibody. A and B, Antirat renin antibody (K026E V). C and D, Localization of both antigens is indicated by brown staining. E, rat prostate section (d 30) incubated without primary antibody. F, A 60-d prostate incubated with primary antibody AT1 (N-10) preabsorbed with specific antigenic peptide.
FIG. 3. Immunocytochemistry of human prostate sections using the anti AT1 monoclonal antibody 6316/G2 (A) and the antihuman renin antibody F37.2D12 (C). Localization of both antigens is indicated by brown staining. As a negative control, slide D was incubated without primary antibody and slide B with primary antibody 6313/G2 preabsorbed with antigenic peptide.
FIG. 4. Upper, Immunoblot illustrating the decrease in AT1 receptor protein expression in d-60 (post puberty) prostate when compared with d 30 (prepuberty). Lower, Histogram representing the percentage of decrease in total band density of AT1 receptor protein expression observed in d-60 animals (n = 9). Band intensity of d-30 prostate was taken as 100%.
Confirmation of the capacity of prostate tissue to synthesize angiotensin was obtained by detection of immunoreactive angiotensin in tissue culture medium from both rat organ cultures (Fig. 5) and from LNCaP cells (Fig. 6). Specificity of the angiotensin II antibody used in this RIA has previously been confirmed by HPLC (30). The data show significant enhancement of immunoreactive angiotensin by DHT in rat prostates (Fig. 5) and by E2 in LNCaP cells. In the latter case, the stimulation of immunoreactive angiotensin by E2 was blocked by the estrogen receptor antagonist, tamoxifen (Fig. 6).
FIG. 5. Secretion of immunoreactive angiotensin II into culture medium by rat prostate in organ culture and the effects of stimulation by DHT (10–8 M) and E2 (10–8 M) for 24 h. Controls received no treatment. Values are nanograms immunoreactive angiotensin per milligram protein. Comparison between control and DHT stimulated, P < 0.05 (Student’s t test, n = 8 throughout).
FIG. 6. Secretion of immunoreactive angiotensin by LNCaP cells. Control media from cells that received no treatment, media from cells that were stimulated for a period of 24 h in the presence of E2 (10–8 M), and media removed from cells stimulated with E2 (10–8 M) and tamoxifen (Tam; 10–6 M). Control vs. E2, P < 0.01 for both assays. Note: Paired Student’s t test, because these are means ± SE (n = 5) of different cell cultures, performed at significantly different times.
Examination of rat prostate before and after puberty illustrated that 30-d-old rats had more intense AT1 receptor expression in the prostate than the sexually mature 60-d-old rats shown by both immunohistochemistry (Fig. 2) and immunoblotting (Fig. 4). In addition, prostates taken from animals maintained on a low-sodium diet showed no change in AT1 expression (Fig. 7). At the same time, adrenal expression of the AT1 receptor, as evaluated by immunoblotting of adrenal protein using the Santa Cruz anti-AT1 antibody, showed that AT1 expression was increased from 0.92 ± 0.10 to 1.94 ± 0.15 densitometry units, consistent with other data on the activation of the circulating RAS-adrenal system previously reported by us and others (33, 34, 37) by this or similar low-sodium diets. However, AT1 expression was significantly enhanced in prostates from animals pretreated with the ACE inhibitor, captopril (Fig. 8).
FIG. 7. A, Immunoblot for AT1 receptor using Santa Cruz antibody N-10 in prostates from control animals and animals given a sodium-deplete diet, with representative histogram (B). No significant effect was observed (n = 6).
FIG. 8. A, Immunoblot of control prostates and those removed from captopril-pretreated animals (n = 6). Captopril pretreatment significantly enhanced AT1 receptor expression. B, Histogram representing data obtained from n = 6 (P < 0.05, Student’s t test).
Discussion
Although traditionally considered a cardiac and renal hormone, there is considerable evidence that angiotensin II is produced throughout the male reproductive tract (14). This is true particularly for the epididymis (20, 38, 39, 40, 41). In addition, the coagulating gland in mice has been shown to be a site of renin expression, which is stimulated by androgen (21, 38). The objective of this study was to investigate the presence of a functional RAS system within human and rat prostate. The results show that the prostate has the potential to synthesize and secrete angiotensin and suggest that it may be a source of the secreted angiotensin that is present in the seminal plasma (30).
The results highlight the existence of a paracrine/autocrine RAS in the rat and human prostate. Evidence was obtained for mRNA for all the components of the RAS in human and rat prostate and within the prostatic cell line LNCaP. Prorenin and AT1 receptor protein was also localized in rat and human prostate. Immunocytochemical staining for (pro)renin showed intense staining in the epithelial region of human prostate, although RT-PCR data showed a relatively low-intensity (pro)renin band. The discrepancy between (pro)renin mRNA and (pro)renin protein expression may arise from various factors; for example, posttranscriptional events or the nonquantitative nature of the PCR method. However, the fact that this tissue RAS was functional was emphasized by secretion of immunoreactive angiotensin from rat organ cultures and the LNCaP cell line.
The results are consistent with, and extend, the finding of others that angiotensin II is present in prostate basal epithelial cells and also the presence of ACE in benign prostatic hyperplasia (42, 43). Together with the localization of renin, the only RAS-specific enzyme of this proteolytic cascade in rat and human prostate epithelial cells, we propose that the evidence is now sufficient to support the identification of a functional local RAS within the prostate.
The presence of angiotensin receptors in prostate has also been shown previously (39, 44); however, Dinh et al. (44) reported that the AT1 receptor was localized predominantly in the periurethral stromal smooth muscle of the human prostate. This contrasts with the presence of AT1 receptor immunopositive staining in glandular epithelium of the human and rat prostate reported in this study. This discrepancy cannot be easily explained. However, both of the antibodies used in the present study, 6313/G2 (Fig. 3), and the Santa Cruz antibody (results not shown) gave the same result. Furthermore, the specificity of immunopositivity was confirmed by preabsorption of the antibody with the specific antigenic peptide in both immunocytochemical and immunoblotting techniques. Epithelial localization of the AT1 receptor was also confirmed through identification of the appropriate mRNA in the LNCaP prostate cell line, which is of epithelial origin. Identification of both AT1 and AT2 receptor mRNA in LNCaP cells has also reported by Lin and Freeman (42).
It is quite clear from this study and others that the prostate itself is also a target for angiotensin, and the paracrine/autocrine role of RAS cannot be overlooked. Expression of AT receptors primarily in the epithelial cells lining the ducts is highly characteristic of many secretory epithelia (12, 13, 44, 45, 46) and perhaps indicates the likelihood that it is involved in regulation of electrolyte transport (40, 47). Angiotensin II has also been associated with noradrenaline release from sympathetic nerves of the rat prostate (39), and the prostatic RAS has been implicated in the pathophysiology of benign prostatic hyperplasia (39, 44, 48). A regulatory role for angiotensin II in prostate stromal growth via the endogenous growth factor heparin-binding EGF-like growth factor has also been reported (42). The intensity of AT1 expression in the prostates of young animals compared with mature adults may suggest that the local importance of angiotensin is greater in young animals, and perhaps this hints at a possible role in prostate development (Figs. 2 and 4).
To examine the regulation of local prostatic RAS, male Wistar rats were maintained on low-sodium diets for a period of 2 weeks, after which time the prostates were removed and the expression of the AT1 receptor examined. It is well recognized that a low-sodium diet results in an enhanced systemic RAS-aldosterone system (34, 37). However, in contrast to the adrenal, the enhanced systemic RAS-aldosterone system brought about by a low-sodium diet had no action on prostate AT1 receptor (Fig. 7).
It is likely that AT1 receptor expression is negatively regulated in the prostate by angiotensin II itself (49) because it is also known to be in various tissues (with the notable exception of the adrenal (50, 51), and this is suggested by the stimulatory action of captopril pretreatment on prostate AT1 expression (Fig. 8).
Hormonal regulation of immunoreactive angiotensin II secretion was examined in vitro from rat prostate cultures and from the LNCaP cell line. The stimulatory action of DHT provides indirect support for local regulation of the RAS. The observation that AT1 receptor expression is lower in mature animals, when testosterone levels are high, than in juveniles, when testosterone is low, suggests that these two may be related, and that it is the high angiotensin output induced by androgen that reduces AT1 receptor in the adult. However, DHT had no effect in LNCaP cells (data not shown), whereas E2 significantly stimulated immunoreactive angiotensin output, an effect that was inhibited by tamoxifen. LNCaP cells, although often used as human prostate model, are in fact carcinoma cells derived from metastasis of supraclavicular lymph node. A distinct aspect of prostate cancer progression is attainment of androgen independence: the ability to function in the absence of DHT (52). The fact that E2 was able to elicit a significant stimulatory effect in the absence of any effect of DHT may be a consequence of cell background and prostate cancer pathology. Complete understanding of the regulation of prostatic RAS in pathological states requires further analysis.
What is clear from this study is that the RAS is regulated in the prostate separately from the systemic RAS (Fig. 7) and in a manner that links it closely with a reproductive function. This is true both for the secretion of angiotensin (Figs. 5 and 6) and for its likely actions, as reflected in receptor expression (Figs. 2 and 4).
In conclusion, the data presented here suggest, although do not prove, that the prostate may be the source of angiotensin II found in seminal plasma. Angiotensin II production maybe regulated by sex steroids, suggesting that when animals become reproductive, angiotensin II will be available for potential stimulation of reproductive functions, for example, the enhancement of sperm motility and capacitation (26, 27, 28, 29).
Acknowledgments
We are grateful to Dr. Dan Bernie (Department of Pathology, St. Bartholomew’s Hospital, London, UK) for help in providing archival prostate tissue.
References
Gupta P, Franco-Saenz R, Mulrow PJ 1995 Locally generated angiotensin-II in the adrenal gland regulates basal, corticotropin-stimulated, and potassium-stimulated aldosterone secretion. Hypertension 25:443–448
Mulrow PJ 1998 Renin-angiotensin system in the adrenal. Horm Metab Res 30:346–349
Vinson GP, Ho MM 1998 The adrenal renin/angiotensin system in the rat. Horm Metab Res 30:355–359
Bader M, Peters J, Baltatu O, Muller DN, Luft FC, Ganten D 2001 Tissue renin-angiotensin systems: new insights from experimental animal models in hypertension research. J Mol Med 79:76–102
Varagic J, Frohlich ED 2002 Local cardiac renin-angiotensin system: hypertension and cardiac failure. J Mol Cell Cardiol 34:1435–1442
MacKenzie SM, Fraser R, Connell JM, Davies E 2002 Local renin-angiotensin systems and their interactions with extra-adrenal corticosteroid production. J Renin Angiotensin Aldosterone Syst 3:214–221
McKinley MJ, Albiston AL, Allen AM, Mathai ML, May CN, McAllen RM, Oldfield BJ, Mendelsohn FA, Chai SY 2003 The brain renin-angiotensin system: location and physiological roles. Int J Biochem Cell Biol 35:901–918
Krege JH, John SWM, Langenbach LL, Hodgin JB, Hagaman JR, Bachman ES, Jennette JC, O’Brien DA, Smithies O 1995 Male-female differences in fertility and blood-pressure in ACE-deficient mice. Nature 375:146–148
Kim HS, Krege JH, Kluckman KD, Hagaman JR, Hodgin JB, Best CF, Jennette JC, Coffman TM, Maeda H, Smithies O 1995 Genetic-control of blood-pressure and the angiotensinogen locus. Proc Natl Acad Sci USA 92:2735–2739
Hagaman JR, Moyer JS, Bachman ES, Siboney M, Magyar PL, Welch JE, Smithies O, Krege JH, O’Brien DA 1998 Angiotensin-converting enzyme and male fertility. Proc Natl Acad Sci USA 95:2552–2557
Sealey JE, Rubattu S 1989 Prorenin and renin as separate mediators of tissue and circulating systems. Am J Hypertens 2:358–366
Vinson GP, Ho MM, Puddefoot JR 1995 The distribution of angiotensin II type 1 receptors, and the tissue renin-angiotensin systems. Mol Med Today 1:35–39
Saridogan E, Djahanbakhch O, Puddefoot JR, Demetroulis C, Collingwood K, Mehta JG, Vinson GP 1996 Angiotensin II receptors and angiotensin II stimulation of ciliary activity in human fallopian tube. J Clin Endocrinol Metab 81:2719–2725
Leung PS, Sernia C 2003 The renin-angiotensin system and male reproduction: new functions for old hormones. J Mol Endocrinol 30:263–270
Jackson B, Cubela RB, Sakaguchi K, Johnston CI 1988 Characterization of angiotensin converting enzyme (ACE) in the testis and assessment of the in vivo effects of the ACE inhibitor perindopril. Endocrinology 123:50–55
Langford KG, Shai SY, Howard TE, Kovac MJ, Overbeek PA, Bernstein KE 1991 Transgenic mice demonstrate a testis-specific promoter for angiotensin-converting enzyme. J Biol Chem 266:15559–15562
Hubert C, Houot AM, Corvol P, Soubrier F 1991 Structure of the angiotensin I-converting enzyme gene. Two alternate promoters correspond to evolutionary steps of a duplicated gene. J Biol Chem 266:15377–15383
Dzau VJ, Brody T, Ellison KE, Pratt RE, Ingelfinger JR 1987 Tissue-specific regulation of renin expression in the mouse. Hypertension 9:III36–III41
Okuyama A, Nonomura N, Koh E, Kondoh N, Takeyama M, Nakamura M, Fujioka H, Matsumoto K, Matsuda M 1988 Induction of renin-angiotensin system in human testis in vivo. Arch Androl 21:29–35
Wong PYD, Uchendu CN 1990 The role of angiotensin-converting enzyme in the rat epididymis. J Endocrinol 125:457–465
Kon Y 1996 Local renin-angiotensin system: especially in coagulating glands of mice. Arch Histol Cytol 59:399–420
Hohlbrugger G, Pschorr J, Dahlheim H 1984 Angiotensin I converting enzyme in the ejaculate of fertile and infertile men. Fertil Steril 41:324–325
Krassnigg F, Niederhauser H, Fink E, Frick J, Schill WB 1989 Angiotensin converting enzyme in human seminal plasma is synthesized by the testis, epididymis and prostate. Int J Androl 12:22–28
Mukhopadhyay AK, Cobilanschi J, Schulze W, Brunswig-Spickenheier B, Leidenberger FA 1995 Human seminal fluid contains significant quantities of prorenin: its correlation with the sperm density. Mol Cell Endocr 109:219–224
Shibahara H, Kamata M, Hu J, Nakagawa H, Obara H, Kondoh N, Shima H, Sato I 2001 Activity of testis angiotensin converting enzyme (ACE) in ejaculated human spermatozoa. Int J Androl 24:295–299
Vinson GP, Puddefoot JR, Ho MM, Barker S, Mehta J, Saridogan E, Djahabakhch O 1995 Type 1 angiotensin II (AT1) receptors in sperm. J Endocrinol 144:369–378
Vinson GP, Mehta J, Evans S, Matthews S, Puddefoot JR, Saridogan E, Holt WV, Djahanbakhch O 1996 Angiotensin-II stimulates sperm motility. Regul Peptides 67:131–135
Fraser LR, Pondel MD, Vinson GP 2001 Calcitonin, angiotensin II and FPP significantly modulate mouse sperm function. Mol Hum Reprod 7:245–253
Fraser LR, Osiguwa OO 2004 Human sperm responses to calcitonin, angiotensin II and fertilization-promoting peptide in prepared semen samples from normal donors and infertility patients. Hum Reprod 19:596–606
O’Mahony OA, Djahanbahkch O, Mahmood T, Puddefoot JR, Vinson GP 2000 Angiotensin II in human seminal fluid. Hum Reprod 15:1345–1349
Barker S, Marchant W, Ho MM, Puddefoot JR, Hinson JP, Clark AJL, Vinson GP 1993 A monoclonal antibody to a conserved sequence in the extracellular domain recognizes the angiotensin II AT1 receptor in mammalian tissues. J Mol Endocrinol 11:241–245
Galen FX, Devaux C, Atlas S, Guyenne T, Menard J, Corvol P, Simon D, Cazaubon C, Richer P, Badouaille G 1984 New monoclonal antibodies directed against human renin. Powerful tools for the investigation of the renin system. J Clin Invest 74:723–735
Aguilera G, Catt KJ 1983 Regulation of aldosterone secretion during altered sodium intake. J Steroid Biochem 19:525–530
Ho MM, Vinson GP 1998 Transcription of (pro)renin mRNA in the rat adrenal cortex, and the effects of ACTH treatment and a low sodium diet. J Endocrinol 157:217–223
Trowell OA 1959 The culture of mature organs in a synthetic medium. Exp Cell Res 16:118–147
Paulson S, Verhage L, Mayer D, Miller K, Schoenhard G 1994 A nonequilibrium radioimmunoassay for angiotensin II. J Pharmacol Toxicol Methods 32:93–97
McEwan PC, Lindop GB, Kenyon CJ 1996 Control of cell-proliferation in the rat adrenal-gland in-vivo by the renin-angiotensin system. Am J Physiol 34:E192–E198
Kon Y, Endoh D, Murakami K, Yamashita T, Watanabe T, Hashimoto Y, Sugimura M 1995 Expression of renin in coagulating glands is regulated by testosterone. Anat Rec 241:451–460
Fabiani ME, Sourial M, Thomas WG, Johnston CI, Frauman AG 2001 Angiotensin II enhances noradrenaline release from sympathetic nerves of the rat prostate via a novel angiotensin receptor: implications for the pathophysiology of benign prostatic hyperplasia. J Endocrinol 171:97–108
Wong PY, Fu WO, Huang SJ, Law WK 1990 Effect of angiotensins on electrogenic anion transport in monolayer cultures of rat epididymis. J Endocrinol 125:449–456
Leung PS, Chan HC, Fu LXM, Leung PY, Chew SBC, Wong PYD 1997 Angiotensin II receptors: localization of type I and type II in rat epididymides of different developmental stages. J Membr Biol 157:97–103
Lin J, Freeman MR 2003 Transactivation of ErbB1 and ErbB2 receptors by angiotensin II in normal human prostate stromal cells. Prostate 54:1–7
Dinh DT, Frauman AG, Sourial M, Casley DJ, Johnston CI, Fabiani ME 2001 Identification, distribution, and expression of angiotensin II receptors in the normal human prostate and benign prostatic hyperplasia. Endocrinology 142:1349–1356
Saridogan E, Djahanbakhch O, Puddefoot JR, Demetroulis C, Dawda R, Hall AJ, Vinson GP 1996 Type 1 angiotensin II receptors in human endometrium. Mol Hum Reprod 2:659–664
Inwang ER, Puddefoot JR, Brown CL, Goode AW, Marsigliante S, Ho MM, Payne JG, Vinson GP 1997 Angiotensin II type 1 receptor expression in human breast tissues. Br J Cancer 75:1279–1283
Vinson GP, Saridogan E, Puddefoot JR, Djahanbakhch O 1997 Tissue renin-angiotensin systems and reproduction. Hum Reprod 12:651–662
Mahmood T, Djahanbakhch O, Burleigh DE, Puddefoot JR, O’Mahony OA, Vinson GP 2002 Effect of angiotensin II on ion transport across human Fallopian tube epithelial cells in vitro. Reproduction 124:573–579
Nassis L, Frauman AG, Ohishi M, Frauman AG, Ohishi M, Zhuo J, Casley J, Casey DJ, Johnston CI, Fabioani ME 2001 Localization of angiotensin-converting enzyme in the human prostate: pathological expression in benign prostatic hyperplasia. J Pathol 195:571–579
Kaschina E, Unger T 2003 Angiotensin AT1/AT2 receptors: regulation, signalling and function. Blood Press 12:70–88
Aguilera G, Hauger RL, Catt KJ 1978 Control of aldosterone secretion during sodium restriction: adrenal receptor regulation and increased adrenal sensitivity to angiotensin II. Proc Natl Acad Sci USA 75:975–979
Aguilera G, Menard RH, Catt KJ 1980 Regulatory actions of angiotensin II on receptors and steroidogenic enzymes in adrenal glomerulosa cells. Endocrinology 107:55–60
Taplin ME, Balk SP 2004 Androgen receptor: a key molecule in the progression of prostate cancer to hormone independence. J Cell Biochem 91:483–490(Orla A. O’Mahony, Stewart)
Address all correspondence and requests for reprints to: Dr. Gavin Vinson, School of Biological Sciences, Queen Mary, University of London, Mile End Road, London E1 4NS, United Kingdom. E-mail: g.p.vinson@qmul.ac.uk.
Abstract
The renin angiotensin system has been shown to have tissue-related functions that are distinct from its systemic roles. We showed that angiotensin II type 1 (AT1) receptors are present in mammalian sperm, and angiotensin II stimulates sperm motility and capacitation. In addition, angiotensin II is present in human seminal plasma at concentrations higher than found in blood. In testing the possibility that the prostate may be the source of seminal plasma angiotensin II, mRNA coding for angiotensinogen, (pro)renin, and angiotensin-converting enzyme were identified by RT-PCR in rat and human prostate and in prostate LNCaP cells, as well as the angiotensin receptors types 1 and 2 (AT1 and AT2) in human tissues and AT1 in rat. In human tissue, immunocytochemistry showed cellular colocalization of renin with the AT1 receptor in secretory epithelial cells. Confirmation of the capacity of the prostate to secrete angiotensin II was shown by the detection of immunoreactive angiotensin in media removed from rat prostate organ cultures and LNCaP cells. Rat prostate angiotensin secretion was enhanced by dihydrotestosterone, but LNCaP angiotensin was stimulated by estradiol. This stimulation was blocked by tamoxifen. Rat prostate AT1 receptor expression was much greater in prepuberal than in postpuberal rats but was not affected by a low-sodium diet. It was, however, significantly enhanced by captopril pretreatment. These findings all suggest the independence of prostate and systemic renin angiotensin system regulation. The data presented here suggest that the prostate may be a source of the secreted angiotensin II found in seminal plasma.
Introduction
IN RECENT YEARS, IT has become evident that the renin angiotensin system (RAS) has a far wider significance than suggested by its traditional roles in water and electrolyte homeostasis. In particular, many tissues have been shown to contain all of the elements of the RAS and can seemingly synthesize the active peptides, particularly angiotensin II, but sometimes including angiotensin III and IV, for purely paracrine activity. These tissues include adrenal, kidney, brain, and the cardiovascular systems (1, 2, 3, 4, 5, 6, 7).
In the reproductive tract, too, there is now considerable evidence that complete function depends on a locally functional RAS. In part, this has been shown by relative infertility in animals with disrupted genes coding for RAS components, such as angiotensin-converting enzyme (ACE) or angiotensinogen (8, 9, 10), but, in addition, the distribution of RAS components, receptors, and associated angiotensin activities demonstrate functions that are exclusively concerned with the reproductive process (11, 12, 13, 14).
In the male, the existence of a specific testis form of ACE has been known for some years (15, 16, 17), whereas RAS components have been described in various tissues, including testis, epididymis, coagulating gland, and prostate (18, 19, 20, 21). Both ACE and prorenin are found in seminal plasma (22, 23, 24, 25). In addition, the presence of angiotensin II type 1 (AT1) receptors in ejaculated rat and human sperm strongly suggested the possibility that angiotensin affects sperm function, and we reported the stimulation by angiotensin II of patterns of motility associated with capacitation in sperm from patients attending a fertility clinic. This action was mediated through the AT1 receptor (26, 27). In vitro, using the hamster oocyte penetration test, angiotensin II has also been shown to increase the oocyte-penetrating ability of both mouse and human sperm (28, 29). Importantly, we have shown that human seminal plasma contains angiotensin II in concentrations higher than in circulating blood plasma (30). Consequently it is appropriate to determine the source of this secreted angiotensin II.
Materials and Methods
Materials
Total human prostate RNA was purchased from Clontech Laboratories Inc. (Palo Alto, CA). LNCaP cells were purchased from the European Collection of Cell Cultures (Salisbury, UK). General laboratory chemicals and reagents were supplied from Sigma-Aldrich (Poole, Dorset UK) unless otherwise indicated. Archival paraffin sections of human trans-urethral prostate resections were obtained from St. Bartholomew’s and the Royal London Hospital Trust with ethical committee approval.
The monoclonal antibody 6313/G2, specific for the AT1 receptor, was generated in our laboratory. This antibody was raised to a synthetic peptide representing residues 8–17 of the N-terminal extracellular domain (31). A second AT1 antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). As described by the manufacturers, this polyclonal antibody, AT1 (N-10), was also raised against a peptide that maps near the amino-terminal of the AT1 receptor. The antihuman (pro)renin antibody (F37.2D12) was a gift from P. Corvol (Institut National de la Santé et de la Recherche Médicale, Paris, France) (32). The rat renin antibody (K 026E V), which was raised against pure mouse submandibular renin, was a gift from Prof. K. Poulsen (Institute for Anatomy and Physiology, Bulowsvej, Denmark). All secondary antibodies were purchased from the Dako Corp. (Carpinteria, CA).
Animal treatments
Male Wistar rats were obtained from commercial sources and maintained at Queen Mary, University of London according to Home Office guidelines. To investigate the regulation of AT1 receptor expression, animal treatments included dietary manipulation and ACE inhibition. Male Wistar rats, 12–14 wk old, were maintained on a diet of whole-meal flour supplemented with 1% Ca2CO3 and 1% NaCl for 2 wk. Low-sodium diets omitted the 1% NaCl. Such dietary sodium regulation of the RAS is well documented and has been used extensively to investigate both paracrine and endocrine regulation of RAS (33, 34). In a second study, captopril (0.5 mg/ml) was added to drinking water for a period of 1 wk. To investigate the potential influence of puberty on the RAS, prostates were removed from six 30-d-old animals (prepuberal) and were compared with those excised from 60-d-old (postpuberal) animals.
Cell culture
Human prostate LNCaP cells were maintained and propagated in RPMI 1640 medium, supplemented with 10% fetal bovine serum, 2 mM glutamine, 1 mM sodium pyruvate, 10 mM nonessential amino acids, 100 U/ml penicillin, and 100 mg/ml streptomycin sulfate.
Rat organ culture
Organ cultures of rat ventral prostate were prepared according to the method of Trowell (35), with some modifications. Rats were killed by stunning and cervical dislocation. Prostates were excised and immediately placed into Krebs-Ringer-HEPES-bicarbonate glucose buffer [130 mM NaCl, 3.2 mM KCl, 1.2 mM CaCl2·H2O, 0.5 mM MgSO4·7H20, 10 mM HEPES, 2 mM NaHCO3, 0.5 mM KH2PO4, and 12 mM glucose (pH 7.4)] with added antibiotics (125 U/ml penicillin, 0.125 mg/ml streptomycin, and 0.05 mg/ml gentomycin) on ice. Once in a sterile class II hood, the prostates were cut into pieces of approximately 1 mm3. Five to seven explants were placed in transwell-clear cell culture inserts (0.4-μm pore size, VWR, Lutterworth, Leicester, UK) and 1 ml phenol red-free medium 199 supplemented with 10% charcoal-stripped fetal bovine serum, penicillin (100 IU/ml), streptomycin sulfate (100 μg/ml), and glutamine (100 μg/ml) was added to each well. All samples were incubated in a humidified atmosphere of 95% O2/5% CO2 for a period of 24 h after which fresh medium was added. Twenty-four hours later, media samples were transferred to tubes containing a mixture of proteinase inhibitors [100 μl, 25 mM phenanthrolene, 125 mM Na2EDTA, 2 g/liter neomycin in 2% (vol/vol) ethanol, 5 mM bestatin, and 2.5 mM chymostatin] (36) and stored at –20 C until required for angiotensin II measurement. Explants were collected into 300 μl radioimmunoprecipitation assay (RIPA) buffer (1x PBS, 0.1% Nonidet P-40, 0.5% sodium deoxycholate, and 0.1% sodium dodecyl sulfate, containing 1 mg/ml phenylmethylsulfonylfluoride, 1 μg/ml aprotinin, and 1 μg/ml soybean trypsin inhibitor), homogenized (3 x 30 sec), and allowed to stand on ice for 30 min. Cellular debris was removed by centrifugation at 10,000 rpm for 10 min at 4 C. Protein content was assayed using the DC method (Bio-Rad Laboratories, Muenchen Germany).
Cell culture for angiotensin II assay
LNCaP cells were seeded into tissue culture flasks (T150 cm2) and, once confluent, serum-free RPMI 1640 medium was added for a period of 48 h. Cells were then stimulated for 24 h with dihydrotestosterone (DHT) (10–8 M) or estradiol (E2) (10–8 M) in the presence of tamoxifen (10–6 M). Media were removed and collected into the previously described inhibitor mix (1 ml) on ice to prevent degradation of angiotensin II. Cells were pelleted by centrifugation at 1000 x g for 5 min, and protein was assayed as above.
Angiotensin II RIA
Media collected from rat prostate cultures and the LNCaP cell line were processed using solid-phase extraction on ENVI-18 columns (Supelco, Poole, UK) as described previously (30) and eluants were lyophilized. Immunoreactive angiotensin II levels were then assayed using an RIA kit supplied by Bachem (UK) Ltd. (St. Helens, Merseyside, UK)
Immunocytochemistry
Archival human prostate sections (n = 12) were obtained from the Department of Morbid Anatomy (The Royal London Hospital, London, UK). Tissues were removed at the time of operation with appropriate consent and placed in 10% formalin for 24 h before routine automated paraffin wax embedding. Sections (8 μm) were mounted on slides, dewaxed in xylene, and rehydrated through graded alcohols. Endogenous peroxidase activity was blocked by 30-min incubation in 3% hydrogen peroxide in methanol. Antigen retrieval was performed by microwave treatment for 10 min in 0.5 M trisodium citrate (pH 6.0). Nonspecific staining was blocked using 0.25% casein in PBS (Dako protein block serum free; Dako Corp.) for 30 min at room temperature followed by incubation with antihuman AT1 receptor antibody (AT1 N-10; Santa Cruz Biotechnology), diluted 1:1000 in PBS supplemented with 5% normal goat serum), the antihuman (pro)renin antibody (dilution 1:750 in PBS) (31), or the AT1 receptor antibody 6313/G2 (1:1000 in PBS) (32) overnight at 4 C. Sections were washed in PBS and subsequently incubated with appropriate biotinylated secondary antibodies (1:200 in PBS) for 30 min at room temperature. Visualization of the antigens was achieved using the peroxidase streptABC complex and incubation with the peroxidase substrate 3',3'-diaminobenzidine. Specificity of the AT1 receptor antibodies was checked by preabsorption of the primary antibody with the appropriate peptide, and, for the (pro)renin antibody, the primary antibody was omitted.
To investigate immunolocalization of the AT1 receptor and renin in rat prostate, tissue was isolated from prepuberal and postpuberal male Wistar rats and fixed in molecular biology fixative (Streck Laboratories. Inc., Omaha, NE) for 16 h at room temperature. Tissues were dehydrated through an alcohol series and embedded in paraffin wax using an automatic tissue processor (Shandon Elliot Scientific Company Ltd., London, UK). Tissue sections were mounted on 3'-aminopropyltriethoxy-silane-coated slides and dried at 37 C overnight.
To prevent endogenous peroxidase activity and to check for nonspecific staining, rat prostate tissues were treated as for human prostate sections. Antigen retrieval was also performed by microwave treatment for 10 min in 0.5 M trisodium citrate (pH 6.0). To identify the AT1 receptor in rat prostate tissue, the same protocols and AT1-specific antibodies were used as for human prostate sections because both the polyclonal AT1 (N-10) and the monoclonal 6313/G2 AT1-specific antibody cross-react with human and rat AT1 receptors. To identify renin in the rat prostate, the rabbit antibody (K 026E V) was used at a 1:500 dilution in PBS at 4 C overnight. Secondary antibody incubation and antigen visualization was as used for human prostate samples.
Immunoblotting
LNCaP cells were grown to confluence, serum starved for 24 h, then stimulated with angiotensin II (10–10 to 10–6 M) for an additional 24 h. Cells were collected into 200 μl RIPA buffer, and total protein was prepared. Rat prostate tissues were collected and homogenized in RIPA buffer on ice. Total protein was prepared, and protein samples were separated on 10% sodium dodecyl sulfate-PAGE gels, incubated for 1.5 h at room temperature in 5% nonfat milk powder in Tris-buffered saline (with 1% Tween), and immunoblotted with AT1 receptor antibody (Santa Cruz Biotechnology) diluted 1:1000 and incubated overnight at 4 C. Immunoreactive bands were detected by enhanced chemiluminescence (Amersham, Hertsfordshire, UK) and exposure to film.
RT-PCR
Total RNA was extracted from confluent LNCaP cells and rat prostate homogenates using the RNeasy minikit (Qiagen Ltd., West Sussex, UK). Human and cell line RNA samples were quantified, and 5 μg of total RNA was added to a mixture of 1 μl of oligo(dT)15 (0.5 mg/ml) with an appropriate volume of ribonuclease-free water and heated to 70 C for 10 min followed by a quick chill on ice. To each tube, 1 μl of dNTP mix (dATP, dTTP, dGTP, and dCTP, each to a final concentration of 10 mM), 2 μl 0.1 M dithiothreitol, and 4 μl 5x first-strand buffer was added and heated to 42 C for a further 2 min. Reverse transcriptase Moloney murine leukemia virus-reverse transcriptase was then added, and each sample was incubated at 42 C for 50 min followed by 70 C for 15 min. PCR was carried out using the primers described in Table 1 at a final concentration of 1 pmol/μl.
TABLE 1. Details of human primers utilized in this study
For investigation of rat tissue samples, the Access RT-PCR system (Promega, Southampton, UK) was used. RT-PCR profiles are outlined in Table 2. PCR cycle number and the RNA concentration were optimized so that linearity was achieved with increasing cycle number and RNA concentration. Optimal conditions are described in Tables 1 and 2. PCR products were separated on 1.5% agarose gels and visualized under UV light.
Statistical analysis
All results are expressed as mean values ± SEM. Differences between control and experimental groups were analyzed using Student’s t test. Statistical significance was defined as P < 0.05.
Results
To confirm the possibility that the prostate might be a potential site for angiotensin II synthesis, evidence for the main RAS components was sought in rat and human prostate tissue and in human prostate cells. Results obtained by RT-PCR are illustrated in Fig. 1. These show that mRNAs for angiotensinogen, renin, ACE, and the AT1 receptor are all transcribed in rat prostate (Fig. 1A), the human prostate (Fig. 1B), and the human prostate epithelial LNCaP cells (Fig. 1C). AT2 mRNA was also identified in all human samples examined. The data are qualitative and not representative of the magnitude of expression of particular components because it was not normalized to a control primer set.
FIG. 1. RT-PCR demonstrated mRNA for all the RAS components in rat (A) and human prostate (B) and LNCaP cells (C). Single bands were obtained throughout (+). Negative control lanes (–) omitted reverse transcriptase. AGT, Angiotensinogen; Ren, renin; AT1 and AT2, AII type 1 and type 2 receptors.
In both the rat prostate (n = 6) (Fig. 2) and the human prostate (n = 12) (Fig. 3), the major site of AT1 receptor immunoactivity was the secretory epithelial cells lining the ducts, with faint reactions in parts of the stroma in human tissue. In human prostate samples, staining intensity did vary among patients, but staining was always observed in both epithelium and the stroma. Confirmation of the epithelial localization of the AT1 receptor was obtained by using a second AT1 receptor antibody as outlined in Materials and Methods. Further evidence for epithelial localization of AT1 receptor was provided by immunoblotting of the LNCaP prostate epithelial cell line protein with an AT1-specific antibody (Santa Cruz Biotechnology), which gave a single band at the appropriate size (data not shown). Evidence for the presence of the AT1 receptor in rat prostate was also obtained by immunoblotting (Fig. 4). Immunostaining of the human prostate using the (pro)renin antibody illustrated that (pro)renin colocalizes with the AT1 receptor in secretory epithelial cells (Fig. 3).
FIG. 2. Immunocytochemistry of 30- and 60- d rat prostate sections using the AT1 (N-10) Santa Cruz polyclonal antibody. A and B, Antirat renin antibody (K026E V). C and D, Localization of both antigens is indicated by brown staining. E, rat prostate section (d 30) incubated without primary antibody. F, A 60-d prostate incubated with primary antibody AT1 (N-10) preabsorbed with specific antigenic peptide.
FIG. 3. Immunocytochemistry of human prostate sections using the anti AT1 monoclonal antibody 6316/G2 (A) and the antihuman renin antibody F37.2D12 (C). Localization of both antigens is indicated by brown staining. As a negative control, slide D was incubated without primary antibody and slide B with primary antibody 6313/G2 preabsorbed with antigenic peptide.
FIG. 4. Upper, Immunoblot illustrating the decrease in AT1 receptor protein expression in d-60 (post puberty) prostate when compared with d 30 (prepuberty). Lower, Histogram representing the percentage of decrease in total band density of AT1 receptor protein expression observed in d-60 animals (n = 9). Band intensity of d-30 prostate was taken as 100%.
Confirmation of the capacity of prostate tissue to synthesize angiotensin was obtained by detection of immunoreactive angiotensin in tissue culture medium from both rat organ cultures (Fig. 5) and from LNCaP cells (Fig. 6). Specificity of the angiotensin II antibody used in this RIA has previously been confirmed by HPLC (30). The data show significant enhancement of immunoreactive angiotensin by DHT in rat prostates (Fig. 5) and by E2 in LNCaP cells. In the latter case, the stimulation of immunoreactive angiotensin by E2 was blocked by the estrogen receptor antagonist, tamoxifen (Fig. 6).
FIG. 5. Secretion of immunoreactive angiotensin II into culture medium by rat prostate in organ culture and the effects of stimulation by DHT (10–8 M) and E2 (10–8 M) for 24 h. Controls received no treatment. Values are nanograms immunoreactive angiotensin per milligram protein. Comparison between control and DHT stimulated, P < 0.05 (Student’s t test, n = 8 throughout).
FIG. 6. Secretion of immunoreactive angiotensin by LNCaP cells. Control media from cells that received no treatment, media from cells that were stimulated for a period of 24 h in the presence of E2 (10–8 M), and media removed from cells stimulated with E2 (10–8 M) and tamoxifen (Tam; 10–6 M). Control vs. E2, P < 0.01 for both assays. Note: Paired Student’s t test, because these are means ± SE (n = 5) of different cell cultures, performed at significantly different times.
Examination of rat prostate before and after puberty illustrated that 30-d-old rats had more intense AT1 receptor expression in the prostate than the sexually mature 60-d-old rats shown by both immunohistochemistry (Fig. 2) and immunoblotting (Fig. 4). In addition, prostates taken from animals maintained on a low-sodium diet showed no change in AT1 expression (Fig. 7). At the same time, adrenal expression of the AT1 receptor, as evaluated by immunoblotting of adrenal protein using the Santa Cruz anti-AT1 antibody, showed that AT1 expression was increased from 0.92 ± 0.10 to 1.94 ± 0.15 densitometry units, consistent with other data on the activation of the circulating RAS-adrenal system previously reported by us and others (33, 34, 37) by this or similar low-sodium diets. However, AT1 expression was significantly enhanced in prostates from animals pretreated with the ACE inhibitor, captopril (Fig. 8).
FIG. 7. A, Immunoblot for AT1 receptor using Santa Cruz antibody N-10 in prostates from control animals and animals given a sodium-deplete diet, with representative histogram (B). No significant effect was observed (n = 6).
FIG. 8. A, Immunoblot of control prostates and those removed from captopril-pretreated animals (n = 6). Captopril pretreatment significantly enhanced AT1 receptor expression. B, Histogram representing data obtained from n = 6 (P < 0.05, Student’s t test).
Discussion
Although traditionally considered a cardiac and renal hormone, there is considerable evidence that angiotensin II is produced throughout the male reproductive tract (14). This is true particularly for the epididymis (20, 38, 39, 40, 41). In addition, the coagulating gland in mice has been shown to be a site of renin expression, which is stimulated by androgen (21, 38). The objective of this study was to investigate the presence of a functional RAS system within human and rat prostate. The results show that the prostate has the potential to synthesize and secrete angiotensin and suggest that it may be a source of the secreted angiotensin that is present in the seminal plasma (30).
The results highlight the existence of a paracrine/autocrine RAS in the rat and human prostate. Evidence was obtained for mRNA for all the components of the RAS in human and rat prostate and within the prostatic cell line LNCaP. Prorenin and AT1 receptor protein was also localized in rat and human prostate. Immunocytochemical staining for (pro)renin showed intense staining in the epithelial region of human prostate, although RT-PCR data showed a relatively low-intensity (pro)renin band. The discrepancy between (pro)renin mRNA and (pro)renin protein expression may arise from various factors; for example, posttranscriptional events or the nonquantitative nature of the PCR method. However, the fact that this tissue RAS was functional was emphasized by secretion of immunoreactive angiotensin from rat organ cultures and the LNCaP cell line.
The results are consistent with, and extend, the finding of others that angiotensin II is present in prostate basal epithelial cells and also the presence of ACE in benign prostatic hyperplasia (42, 43). Together with the localization of renin, the only RAS-specific enzyme of this proteolytic cascade in rat and human prostate epithelial cells, we propose that the evidence is now sufficient to support the identification of a functional local RAS within the prostate.
The presence of angiotensin receptors in prostate has also been shown previously (39, 44); however, Dinh et al. (44) reported that the AT1 receptor was localized predominantly in the periurethral stromal smooth muscle of the human prostate. This contrasts with the presence of AT1 receptor immunopositive staining in glandular epithelium of the human and rat prostate reported in this study. This discrepancy cannot be easily explained. However, both of the antibodies used in the present study, 6313/G2 (Fig. 3), and the Santa Cruz antibody (results not shown) gave the same result. Furthermore, the specificity of immunopositivity was confirmed by preabsorption of the antibody with the specific antigenic peptide in both immunocytochemical and immunoblotting techniques. Epithelial localization of the AT1 receptor was also confirmed through identification of the appropriate mRNA in the LNCaP prostate cell line, which is of epithelial origin. Identification of both AT1 and AT2 receptor mRNA in LNCaP cells has also reported by Lin and Freeman (42).
It is quite clear from this study and others that the prostate itself is also a target for angiotensin, and the paracrine/autocrine role of RAS cannot be overlooked. Expression of AT receptors primarily in the epithelial cells lining the ducts is highly characteristic of many secretory epithelia (12, 13, 44, 45, 46) and perhaps indicates the likelihood that it is involved in regulation of electrolyte transport (40, 47). Angiotensin II has also been associated with noradrenaline release from sympathetic nerves of the rat prostate (39), and the prostatic RAS has been implicated in the pathophysiology of benign prostatic hyperplasia (39, 44, 48). A regulatory role for angiotensin II in prostate stromal growth via the endogenous growth factor heparin-binding EGF-like growth factor has also been reported (42). The intensity of AT1 expression in the prostates of young animals compared with mature adults may suggest that the local importance of angiotensin is greater in young animals, and perhaps this hints at a possible role in prostate development (Figs. 2 and 4).
To examine the regulation of local prostatic RAS, male Wistar rats were maintained on low-sodium diets for a period of 2 weeks, after which time the prostates were removed and the expression of the AT1 receptor examined. It is well recognized that a low-sodium diet results in an enhanced systemic RAS-aldosterone system (34, 37). However, in contrast to the adrenal, the enhanced systemic RAS-aldosterone system brought about by a low-sodium diet had no action on prostate AT1 receptor (Fig. 7).
It is likely that AT1 receptor expression is negatively regulated in the prostate by angiotensin II itself (49) because it is also known to be in various tissues (with the notable exception of the adrenal (50, 51), and this is suggested by the stimulatory action of captopril pretreatment on prostate AT1 expression (Fig. 8).
Hormonal regulation of immunoreactive angiotensin II secretion was examined in vitro from rat prostate cultures and from the LNCaP cell line. The stimulatory action of DHT provides indirect support for local regulation of the RAS. The observation that AT1 receptor expression is lower in mature animals, when testosterone levels are high, than in juveniles, when testosterone is low, suggests that these two may be related, and that it is the high angiotensin output induced by androgen that reduces AT1 receptor in the adult. However, DHT had no effect in LNCaP cells (data not shown), whereas E2 significantly stimulated immunoreactive angiotensin output, an effect that was inhibited by tamoxifen. LNCaP cells, although often used as human prostate model, are in fact carcinoma cells derived from metastasis of supraclavicular lymph node. A distinct aspect of prostate cancer progression is attainment of androgen independence: the ability to function in the absence of DHT (52). The fact that E2 was able to elicit a significant stimulatory effect in the absence of any effect of DHT may be a consequence of cell background and prostate cancer pathology. Complete understanding of the regulation of prostatic RAS in pathological states requires further analysis.
What is clear from this study is that the RAS is regulated in the prostate separately from the systemic RAS (Fig. 7) and in a manner that links it closely with a reproductive function. This is true both for the secretion of angiotensin (Figs. 5 and 6) and for its likely actions, as reflected in receptor expression (Figs. 2 and 4).
In conclusion, the data presented here suggest, although do not prove, that the prostate may be the source of angiotensin II found in seminal plasma. Angiotensin II production maybe regulated by sex steroids, suggesting that when animals become reproductive, angiotensin II will be available for potential stimulation of reproductive functions, for example, the enhancement of sperm motility and capacitation (26, 27, 28, 29).
Acknowledgments
We are grateful to Dr. Dan Bernie (Department of Pathology, St. Bartholomew’s Hospital, London, UK) for help in providing archival prostate tissue.
References
Gupta P, Franco-Saenz R, Mulrow PJ 1995 Locally generated angiotensin-II in the adrenal gland regulates basal, corticotropin-stimulated, and potassium-stimulated aldosterone secretion. Hypertension 25:443–448
Mulrow PJ 1998 Renin-angiotensin system in the adrenal. Horm Metab Res 30:346–349
Vinson GP, Ho MM 1998 The adrenal renin/angiotensin system in the rat. Horm Metab Res 30:355–359
Bader M, Peters J, Baltatu O, Muller DN, Luft FC, Ganten D 2001 Tissue renin-angiotensin systems: new insights from experimental animal models in hypertension research. J Mol Med 79:76–102
Varagic J, Frohlich ED 2002 Local cardiac renin-angiotensin system: hypertension and cardiac failure. J Mol Cell Cardiol 34:1435–1442
MacKenzie SM, Fraser R, Connell JM, Davies E 2002 Local renin-angiotensin systems and their interactions with extra-adrenal corticosteroid production. J Renin Angiotensin Aldosterone Syst 3:214–221
McKinley MJ, Albiston AL, Allen AM, Mathai ML, May CN, McAllen RM, Oldfield BJ, Mendelsohn FA, Chai SY 2003 The brain renin-angiotensin system: location and physiological roles. Int J Biochem Cell Biol 35:901–918
Krege JH, John SWM, Langenbach LL, Hodgin JB, Hagaman JR, Bachman ES, Jennette JC, O’Brien DA, Smithies O 1995 Male-female differences in fertility and blood-pressure in ACE-deficient mice. Nature 375:146–148
Kim HS, Krege JH, Kluckman KD, Hagaman JR, Hodgin JB, Best CF, Jennette JC, Coffman TM, Maeda H, Smithies O 1995 Genetic-control of blood-pressure and the angiotensinogen locus. Proc Natl Acad Sci USA 92:2735–2739
Hagaman JR, Moyer JS, Bachman ES, Siboney M, Magyar PL, Welch JE, Smithies O, Krege JH, O’Brien DA 1998 Angiotensin-converting enzyme and male fertility. Proc Natl Acad Sci USA 95:2552–2557
Sealey JE, Rubattu S 1989 Prorenin and renin as separate mediators of tissue and circulating systems. Am J Hypertens 2:358–366
Vinson GP, Ho MM, Puddefoot JR 1995 The distribution of angiotensin II type 1 receptors, and the tissue renin-angiotensin systems. Mol Med Today 1:35–39
Saridogan E, Djahanbakhch O, Puddefoot JR, Demetroulis C, Collingwood K, Mehta JG, Vinson GP 1996 Angiotensin II receptors and angiotensin II stimulation of ciliary activity in human fallopian tube. J Clin Endocrinol Metab 81:2719–2725
Leung PS, Sernia C 2003 The renin-angiotensin system and male reproduction: new functions for old hormones. J Mol Endocrinol 30:263–270
Jackson B, Cubela RB, Sakaguchi K, Johnston CI 1988 Characterization of angiotensin converting enzyme (ACE) in the testis and assessment of the in vivo effects of the ACE inhibitor perindopril. Endocrinology 123:50–55
Langford KG, Shai SY, Howard TE, Kovac MJ, Overbeek PA, Bernstein KE 1991 Transgenic mice demonstrate a testis-specific promoter for angiotensin-converting enzyme. J Biol Chem 266:15559–15562
Hubert C, Houot AM, Corvol P, Soubrier F 1991 Structure of the angiotensin I-converting enzyme gene. Two alternate promoters correspond to evolutionary steps of a duplicated gene. J Biol Chem 266:15377–15383
Dzau VJ, Brody T, Ellison KE, Pratt RE, Ingelfinger JR 1987 Tissue-specific regulation of renin expression in the mouse. Hypertension 9:III36–III41
Okuyama A, Nonomura N, Koh E, Kondoh N, Takeyama M, Nakamura M, Fujioka H, Matsumoto K, Matsuda M 1988 Induction of renin-angiotensin system in human testis in vivo. Arch Androl 21:29–35
Wong PYD, Uchendu CN 1990 The role of angiotensin-converting enzyme in the rat epididymis. J Endocrinol 125:457–465
Kon Y 1996 Local renin-angiotensin system: especially in coagulating glands of mice. Arch Histol Cytol 59:399–420
Hohlbrugger G, Pschorr J, Dahlheim H 1984 Angiotensin I converting enzyme in the ejaculate of fertile and infertile men. Fertil Steril 41:324–325
Krassnigg F, Niederhauser H, Fink E, Frick J, Schill WB 1989 Angiotensin converting enzyme in human seminal plasma is synthesized by the testis, epididymis and prostate. Int J Androl 12:22–28
Mukhopadhyay AK, Cobilanschi J, Schulze W, Brunswig-Spickenheier B, Leidenberger FA 1995 Human seminal fluid contains significant quantities of prorenin: its correlation with the sperm density. Mol Cell Endocr 109:219–224
Shibahara H, Kamata M, Hu J, Nakagawa H, Obara H, Kondoh N, Shima H, Sato I 2001 Activity of testis angiotensin converting enzyme (ACE) in ejaculated human spermatozoa. Int J Androl 24:295–299
Vinson GP, Puddefoot JR, Ho MM, Barker S, Mehta J, Saridogan E, Djahabakhch O 1995 Type 1 angiotensin II (AT1) receptors in sperm. J Endocrinol 144:369–378
Vinson GP, Mehta J, Evans S, Matthews S, Puddefoot JR, Saridogan E, Holt WV, Djahanbakhch O 1996 Angiotensin-II stimulates sperm motility. Regul Peptides 67:131–135
Fraser LR, Pondel MD, Vinson GP 2001 Calcitonin, angiotensin II and FPP significantly modulate mouse sperm function. Mol Hum Reprod 7:245–253
Fraser LR, Osiguwa OO 2004 Human sperm responses to calcitonin, angiotensin II and fertilization-promoting peptide in prepared semen samples from normal donors and infertility patients. Hum Reprod 19:596–606
O’Mahony OA, Djahanbahkch O, Mahmood T, Puddefoot JR, Vinson GP 2000 Angiotensin II in human seminal fluid. Hum Reprod 15:1345–1349
Barker S, Marchant W, Ho MM, Puddefoot JR, Hinson JP, Clark AJL, Vinson GP 1993 A monoclonal antibody to a conserved sequence in the extracellular domain recognizes the angiotensin II AT1 receptor in mammalian tissues. J Mol Endocrinol 11:241–245
Galen FX, Devaux C, Atlas S, Guyenne T, Menard J, Corvol P, Simon D, Cazaubon C, Richer P, Badouaille G 1984 New monoclonal antibodies directed against human renin. Powerful tools for the investigation of the renin system. J Clin Invest 74:723–735
Aguilera G, Catt KJ 1983 Regulation of aldosterone secretion during altered sodium intake. J Steroid Biochem 19:525–530
Ho MM, Vinson GP 1998 Transcription of (pro)renin mRNA in the rat adrenal cortex, and the effects of ACTH treatment and a low sodium diet. J Endocrinol 157:217–223
Trowell OA 1959 The culture of mature organs in a synthetic medium. Exp Cell Res 16:118–147
Paulson S, Verhage L, Mayer D, Miller K, Schoenhard G 1994 A nonequilibrium radioimmunoassay for angiotensin II. J Pharmacol Toxicol Methods 32:93–97
McEwan PC, Lindop GB, Kenyon CJ 1996 Control of cell-proliferation in the rat adrenal-gland in-vivo by the renin-angiotensin system. Am J Physiol 34:E192–E198
Kon Y, Endoh D, Murakami K, Yamashita T, Watanabe T, Hashimoto Y, Sugimura M 1995 Expression of renin in coagulating glands is regulated by testosterone. Anat Rec 241:451–460
Fabiani ME, Sourial M, Thomas WG, Johnston CI, Frauman AG 2001 Angiotensin II enhances noradrenaline release from sympathetic nerves of the rat prostate via a novel angiotensin receptor: implications for the pathophysiology of benign prostatic hyperplasia. J Endocrinol 171:97–108
Wong PY, Fu WO, Huang SJ, Law WK 1990 Effect of angiotensins on electrogenic anion transport in monolayer cultures of rat epididymis. J Endocrinol 125:449–456
Leung PS, Chan HC, Fu LXM, Leung PY, Chew SBC, Wong PYD 1997 Angiotensin II receptors: localization of type I and type II in rat epididymides of different developmental stages. J Membr Biol 157:97–103
Lin J, Freeman MR 2003 Transactivation of ErbB1 and ErbB2 receptors by angiotensin II in normal human prostate stromal cells. Prostate 54:1–7
Dinh DT, Frauman AG, Sourial M, Casley DJ, Johnston CI, Fabiani ME 2001 Identification, distribution, and expression of angiotensin II receptors in the normal human prostate and benign prostatic hyperplasia. Endocrinology 142:1349–1356
Saridogan E, Djahanbakhch O, Puddefoot JR, Demetroulis C, Dawda R, Hall AJ, Vinson GP 1996 Type 1 angiotensin II receptors in human endometrium. Mol Hum Reprod 2:659–664
Inwang ER, Puddefoot JR, Brown CL, Goode AW, Marsigliante S, Ho MM, Payne JG, Vinson GP 1997 Angiotensin II type 1 receptor expression in human breast tissues. Br J Cancer 75:1279–1283
Vinson GP, Saridogan E, Puddefoot JR, Djahanbakhch O 1997 Tissue renin-angiotensin systems and reproduction. Hum Reprod 12:651–662
Mahmood T, Djahanbakhch O, Burleigh DE, Puddefoot JR, O’Mahony OA, Vinson GP 2002 Effect of angiotensin II on ion transport across human Fallopian tube epithelial cells in vitro. Reproduction 124:573–579
Nassis L, Frauman AG, Ohishi M, Frauman AG, Ohishi M, Zhuo J, Casley J, Casey DJ, Johnston CI, Fabioani ME 2001 Localization of angiotensin-converting enzyme in the human prostate: pathological expression in benign prostatic hyperplasia. J Pathol 195:571–579
Kaschina E, Unger T 2003 Angiotensin AT1/AT2 receptors: regulation, signalling and function. Blood Press 12:70–88
Aguilera G, Hauger RL, Catt KJ 1978 Control of aldosterone secretion during sodium restriction: adrenal receptor regulation and increased adrenal sensitivity to angiotensin II. Proc Natl Acad Sci USA 75:975–979
Aguilera G, Menard RH, Catt KJ 1980 Regulatory actions of angiotensin II on receptors and steroidogenic enzymes in adrenal glomerulosa cells. Endocrinology 107:55–60
Taplin ME, Balk SP 2004 Androgen receptor: a key molecule in the progression of prostate cancer to hormone independence. J Cell Biochem 91:483–490(Orla A. O’Mahony, Stewart)