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Endocrinology Vol. 142, No. 3 1349-1356
Copyright © 2001 by The Endocrine Society

ARTICLES

Identification, Distribution, and Expression of Angiotensin II Receptors in the Normal Human Prostate and Benign Prostatic Hyperplasia1

Diem T. Dinh2, Albert G. Frauman, Melissa Sourial, David J. Casley, Colin I. Johnston and Maurice E. Fabiani

Department of Medicine and Clinical Pharmacology and Therapeutics Unit (A.G.F.), University of Melbourne, Austin and Repatriation Medical Centre, Heidelberg, Victoria 3084, Australia

Address all correspondence and requests for reprints to: Dr. Maurice E. Fabiani, Department of Medicine, University of Melbourne, Austin and Repatriation Medical Centre, Heidelberg, Victoria 3084, Australia. E-mail: m.fabiani{at}austin.unimelb.edu.au


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The tissue distribution, cellular localization, and level of expression of angiotensin II (Ang II) receptors were examined in the normal human prostate and benign prostatic hyperplasia (BPH) by in vitro autoradiography, immunohistochemistry, and radioligand binding studies. In the normal human prostate, Ang II receptors were of the AT1 subtype and localized predominantly to periurethral stromal smooth muscle. The AT1 receptor antagonist losartan totally displaced specific [125I]-[Sar1,Ile8]Ang II binding, in a concentration-dependent manner, whereas the AT2 receptor antagonist PD123319 was without effect. There was no significant difference in receptor affinity, but AT1 receptor density was markedly reduced in BPH compared with that in normal prostate. In rat prostate, Ang II (0.01–1 µM) produced a concentration-dependent increase in [3H]-noradrenaline release from sympathetic nerves. The findings of the present study suggest that angiotensin AT1 receptors predominate in the human prostate. The high concentration of AT1 receptors in the periurethral region suggests a role for Ang II in modulating cell growth, smooth muscle tone, and possibly micturition. Furthermore, down-regulation of AT1 receptors in BPH may be due to receptor hyperstimulation by increased local levels of Ang II in BPH. Finally, Ang II may play a functional role in modulating sympathetic transmission in the prostate. These data support the novel concept that activation of the renin-angiotensin system may be involved in the pathophysiology of BPH.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
BENIGN PROSTATIC hyperplasia (BPH) represents the most common cause of urinary obstruction affecting men past middle age and is frequently associated with hypertension (1, 2). BPH is characterized by an increase in cellular growth and sympathetic tone within the prostate (3). The renin-angiotensin system (RAS) functions as both a circulating system and a local tissue autocrine/paracrine system and has long been implicated in the development of hypertension and cardiovascular growth and remodeling (4). The potent octapeptide angiotensin II (Ang II) is the principal mediator of the RAS and a powerful stimulator of cell growth and sympathetic neuroeffector function, and promotes left ventricular and vascular hypertrophy (5, 6, 7, 8). Ang II interacts with at least two receptors, namely AT1 and AT2. Most of the well described actions of Ang II, such as vasoconstriction, stimulation of cellular proliferation, and facilitation of sympathetic transmission, are mediated by the AT1 receptor (9, 10). The role of the AT2 receptor is not fully understood, but recent studies suggest that it may be involved in antiproliferation, cellular differentiation, and apoptosis (9, 10, 11).

Angiotensin-converting enzyme (ACE) is a major component of the RAS that is ultimately responsible for the production of Ang II. It has been reported, albeit some time ago, that the biochemical activity of ACE in the prostate is markedly enhanced in BPH (12, 13). Little, if any, work has since been undertaken to examine the role of the RAS in the human prostate under normal or pathophysiological conditions. More recently, our group has demonstrated that both messenger RNA and protein expression of ACE are also enhanced in BPH (14, 15). It is likely therefore that local prostatic levels of Ang II are elevated in BPH, which may influence cell growth and/or smooth muscle tone via Ang II receptors. Hyperactivity of the RAS may thus represent an important factor in the pathophysiology of BPH as well as hypertension. Nonetheless, nothing is known about Ang II receptors in the human prostate. Thus, the present study was undertaken to determine the tissue distribution and cellular localization of Ang II receptors in the human prostate by quantitative in vitro autoradiography and immunohistochemistry, and to establish whether there are any differences in BPH. In addition, radioligand binding studies were performed to determine the binding properties of Ang II receptors in normal prostate and BPH membranes. Finally, we also examined the effects of Ang II on [3H]-noradrenaline release from the rat prostate to ascribe a possible functional role of Ang II within the prostate.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Tissue preparation
Human prostate tissues were obtained from post-mortem subjects with no known history of ACE inhibitor or AT1 receptor antagonist usage. Normal and hypertrophied/hyperplastic prostates (BPH) were characterized and confirmed histologically by an expert pathologist (Department of Anatomical Pathology, Austin and Repatriation Medical Centre). The use of human prostate tissues was approved by the Austin and Repatriation Medical Centre Human Ethics Committee. The collected tissues were snap-frozen in isopentane-dry ice (-40 C) and stored at -80 C until required for use.

Quantitative in vitro autoradiography
Tissue sections of 20 µm thickness were cut on a Microm HM505E cryostat (Microm GmbH, Walldorf, Germany) at -20 C and dehydrated overnight under reduced pressure at 4 C. The methods for in vitro autoradiographic localization of Ang II receptors with [125I]-[Sar1,Ile8]Ang II have been described previously (16, 17, 18). Briefly, tissue sections were incubated in sodium phosphate buffer (pH 7.4) containing 10 mM Na2HPO4, 150 mM NaCl, 5 mM Na2EDTA, 0.2% BSA, and 0.4 mM bacitracin with 0.5 nM [125I]-[Sar1,Ile8]Ang II, for 2 h at room temperature. The identification of Ang II receptor subtypes was determined by the presence of an excess (10 µM) of the Ang II receptor antagonists losartan, irbesartan, and candesartan (AT1 selective) and PD123319 (AT2 selective). Nonspecific binding was determined in the presence of an excess (1 µM) of unlabeled Ang II amide. After incubation, tissue sections were washed with ice-cold buffer to remove nonspecifically bound radioligand. The sections were dried, loaded into x-ray cassettes together with a set of radioactivity standards, and exposed to AGFA Curix Ortho HT-G x-ray films (Agfa-Gevaert, Mortsel, Belgium) for 2 weeks. Serial sections were stained with hematoxylin and eosin and examined with autoradiographs for the anatomical localization of [125I]-[Sar1,Ile8]Ang II binding.

Emulsion light microscopic autoradiography
After radioligand incubation and washing (see above), consecutive tissue sections (10 µm) were fixed in paraformaldehyde vapor for 2 days, dipped into LM-1 liquid emulsion (Amersham Pharmacia Biotech, Amersham, Buckinghamshire, UK), and stored in a light-proof box with silica gel for 2 weeks at 4 C. After exposure, slides were processed with Kodak 19 developer (Eastman Kodak Co., Rochester, NY) and stained with hematoxylin and eosin before being examined histologically by light microscopy for the cellular localization of Ang II receptors.

Quantification
Both macroscopic and microscopic autoradiographs were quantified using a MCID image analysis system (Imaging Research, Inc., Toronto, Canada). To obtain quantitative data on AT1 receptor binding within the periurethral region of the prostate, up to 20 samples were randomly selected from the autoradiographs, and the values are expressed as disintegrations per min/mm2 (macroscopic autoradiographs) and grains/mm2 (emulsion slides). The validity of this method for quantitation has been previously established (17, 18). Specific binding was calculated by subtracting nonspecific binding from total binding.

Preparation of prostate membranes
The periurethral and lateral regions of normal prostate and BPH specimens were dissected out and homogenized using a Polytron tissue homogenizer (Kinematica Disperser Polytron PT3000, Littau, Switzerland) in a buffer containing 50 mM Tris-HCl, 0.5 mM EDTA, 0.1 mM phenylmethylsulfonylfluoride, 0.01% bacitracin, and 4 µg/ml leupeptin (pH 7.4). The homogenate was centrifuged (600 x g, 5 min), and the supernatant was collected and then recentrifuged (23,000 x g, 20 min). The pellet was resuspended in the buffer solution and recentrifuged (23,000 x g, 20 min). The final membrane pellet was resuspended in the same buffer but without bacitracin and leupeptin. All steps were performed at 4 C. Prostatic membranes were prepared immediately before binding studies were performed (see below). The protein concentration was measured by the Bradford method using BSA as standard (19).

[125I]-[Sar1,Ile8]Ang II binding studies
Prostate membranes (70 µg) were added to duplicate tubes and incubated in a buffer solution (50 mM Tris-HCl, 10 mM MgCl2, 0.1 mM phenylmethylsulfonylfluoride, 0.01% bacitracin, 1 µg/ml leupeptin, and 0.1% BSA, pH 7.4) with various concentrations of [125I]-[Sar1,Ile8]Ang II (0.05–10 nM) for saturation analysis (1 nM for competition studies) in the absence or presence of the nonpeptide Ang II receptor antagonists losartan (AT1 selective) and PD123319 (AT2 selective), in the concentration range of 1 x 10-11 to 1 x 10-5 M and in a final assay volume of 250 µl. This incubation was performed for 1 h at 25 C and terminated by rapid filtration through GF/C filters (Whatman, Maidstone, UK) soaked in 0.1% (vol/vol) polyethyleimine/dH2O, followed by washing (four times, 4 ml) with ice-cold buffer (50 mM Tris-HCl and 150 mM NaCl, pH 7.4), using a Brandel automatic filtration apparatus (Biomedical Research and Development Laboratories, Inc., Gaithersburg, MD). Tissue-bound radioactivity was measured using a {gamma}-radiation counter (1260 Multigamma II, LKB Wallac Inc., Turku, Finland). Specific [125I]-[Sar1,Ile8]Ang II binding was calculated in the presence of 10 µM unlabeled Ang II amide. Data were analyzed using the nonlinear regression program LIGAND (20).

Immunohistochemistry
Sections (4 µm) of formalin-fixed, paraffin-embedded prostate tissues were mounted on 2% silanized slides. Antigen retrieval was performed using 10 mg protease VIII (Sigma, St. Louis, MO) in PBS (pH 7.2) for 10 min at 37 C, and sections were treated with 3% H2O2-water for 5 min to remove endogenous peroxidase activity. Immunostaining was carried out using the immunoperoxidase method (Catalyzed Signal Amplification System kit, DAKO Corp., Carpinteria, CA). The serum-free protein was applied on tissue sections for 5 min and incubated with antihuman mouse monoclonal antibodies for the detection of smooth muscle {alpha}-actin (Zymed Laboratories Inc., South San Francisco, CA) and AT1 receptors (6313/G2 antibody diluted 1:10 in PBS for 3 h at room temperature; a gift from Prof. G. P. Vinson, Division of Biomedical Sciences, St Bartholomew’s and Royal London School of Medicine and Dentistry, Queen Mary and Westfield College, London, UK). After the primary antibody step, sections were sequentially incubated at room temperature for 15 min with biotinylated rabbit antimouse antibody, streptavidin-biotin complex, amplification reagent, and streptavidin-peroxidase. Finally, immunoreactivity was visualized by the addition of diaminobenzidine. The sections were counterstained with hematoxylin and mounted in aqueous medium before histological examination. The specificity of this AT1 receptor antibody has been validated and confirmed previously (21, 22, 23, 24). Furthermore, appropriate negative controls were tested on adjacent sections by substituting the primary antibody with PBS and normal nonimmune mouse serum.

[3H]-Noradrenaline release studies
The experimental protocol for the use of rat prostates was approved by the Austin and Repatriation Medical Centre Animal Ethics Committee and complied with the National Health and Medical Research Council of Australia guidelines for animal experimentation. Adult male Sprague Dawley rats (250–350 g) were killed by decapitation, and the prostates were removed. Each prostate tissue was initially placed in a glass-jacketed organ bath containing 2 ml physiological salt solution (PSS), continuously gassed with carbogen (95% O2-5% CO2), and maintained at 37 C. The composition of the PSS was 118 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl2, 0.45 mM MgSO4, 25 mM NaHCO3, 1.03 mM KH2PO4, 11.1 mM D-(+)-glucose, 0.067 mM EDTA, and 0.14 mM ascorbic acid. The noradrenergic transmitter stores of the rat prostate were radiolabeled, as described previously for other sympathetically innervated tissues (25, 26, 27), by incubating the tissues with [3H]-noradrenaline (SA, 30–50 Ci/mmol) for 30 min. After radiolabeling, each prostate preparation was rinsed in a small volume of PSS and then mounted between two platinum wire electrodes in an acrylic flow cell and superfused with PSS at a rate of 2 ml/min using a peristaltic pump (Wiz, ISCO, Lincoln, NE). To remove loosely bound radioactivity, each prostate preparation was superfused with PSS for 90 min before experimental procedures were initiated. After the first 30 min of this washout period, a priming stimulus was applied for 30 sec (1-msec pulses, 5 Hz, 15 V) to further assist in the removal of nonspecifically bound radioactive material. After washout, the intrinsic sympathetic nerves of the prostate preparations were subjected to two 60-sec periods of electrical field stimulation (1 msec, 5 Hz, 15 V), delivered 30 min apart. The effects of Ang II on stimulation-induced (S-I) [3H]-noradrenaline release were examined by introducing the peptide 15 min before the second period of stimulation, which then remained present for the remainder of the experiment. The superfusate emanating from the prostate preparations was collected at 3-min intervals by an automated fraction collector (ISCO Retriever IV). Each 3-min (6-ml) fraction of superfusate was mixed with 4 ml Ultima Gold (Packard Instrument Co., Meriden, CT), and the radioactive content was measured by liquid scintillation counting. Corrections for counting efficiency were made by external automated standardization, and the data were expressed as disintegrations per min.

Drugs, radioligands, and materials
[Sar1,Ile8]Ang II and Ang II amide were purchased from Peninsula Laboratories Inc. (Belmont, CA). Losartan, irbesartan, and candesartan were provided by Merck, Sharp & Dohme (Sydney, New South Wales, Australia), Bristol-Myers Squibb Co. (Princeton, NJ), and Astra H|$$|Adassle AB (Molndal, Sweden). PD123319 was purchased from Research Biochemicals International (Natick, MA). All other chemicals were obtained from Sigma. The antagonist analog of Ang II, [Sar1,Ile8]Ang II, was radioiodinated using the chloramine-T method and was purified by HPLC (28). [3H]Noradrenaline was obtained from Amersham Pharmacia Biotech.

Statistical analysis
Quantitative data are expressed as the mean ± SEM. Where appropriate, the data were analyzed by either one-way ANOVA followed by Dunnett’s test or unpaired Student’s t test. P < 0.05 was taken to indicate statistical significance.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Autoradiographic localization of Ang II receptors in human prostate
Figure 1Go shows computer-generated color autoradiographs of Ang II receptor binding in the normal human prostate from a 51-yr-old man and in a similarly aged 56-yr-old patient with BPH using the radioligand [125I]-[Sar1,Ile8]Ang II. In all tissues studied (n = 8), Ang II receptor binding in the human prostate was predominantly of the AT1 receptor subtype (Fig. 1AGo); an excess of the AT1 receptor antagonist losartan (10 µM) abolished Ang II receptor binding (Fig. 1BGo), whereas an excess of the AT2 receptor antagonist PD123319 (10 µM) was without effect (Fig. 1AGo). The effects of other AT1 receptor antagonists, such as irbesartan and candesartan, were also tested and yielded results identical to those obtained with losartan (data not shown). Nonspecific binding, as determined by the presence of an excess of unlabeled Ang II amide (1 µM), was very low (data not shown) and comparable to the level of binding observed in the presence of losartan (Fig. 1Go, B and D). In the normal human prostate, AT1 receptor binding was confined to the periurethral region (Fig. 1AGo). When film autoradiographs (n = 8 for each group) were compared with their corresponding hematoxylin and eosin sections, AT1 receptor binding was localized to stromal smooth muscle. This pattern of AT1 receptor binding was observed in all normal prostate sections regardless of age. As shown in Fig. 1Go, A and B, and quantitatively in Fig. 3AGo, the density of AT1 receptors was markedly reduced in BPH compared with the normal prostate [specific binding: 886 ± 120 dpm/mm2 (n = 8) vs. 3,275 ± 140 dpm/mm2 (n = 8), respectively; P < 0.001]. The emulsion light microscopic localization of AT1 receptors in the human prostate is shown in Fig. 2Go, A and B, and appeared to be localized to stromal smooth muscle. AT1 receptors were again significantly decreased in the periurethral region in BPH compared with the normal prostate [5.2 ± 0.4 grains/mm2 (n = 3) vs. 13.1 ± 0.2 grains/mm2 (n = 3), respectively; P < 0.001; Fig. 3BGo].



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  		Figure 1. Autoradiographic mapping of Ang II receptors in whole-mounted sections of the human prostate. A, AT1 receptor binding in normal prostate (51-yr-old man). B, AT2 receptor binding in normal prostate. C, AT1 receptor binding in BPH (56-yr-old man). D, AT2 receptor binding in BPH. AT1 receptor binding was defined as that remaining in the presence of an excess (10 µM) of the AT2-selective antagonist PD1233319, whereas AT2 receptor binding was defined as that remaining in the presence of an excess (10 µM) of the AT1-selective antagonist losartan. Red represents high density binding sites, whereas blue/green represents low density binding or background levels. High levels of AT1 receptor binding were demonstrated in the periurethral region (PU) of the normal human prostate, which were markedly reduced in BPH.

 


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  		Figure 3. Quantitative values of AT1 receptor density in the periurethral region in normal human prostate and BPH. A, Macroscopic autoradiographs (disintegrations per min/mm2); B, microscopic emulsion autoradiographs (grains/mm2). Each column represents the mean ± SEM, the number of tissues is indicated in parentheses. *, Significantly different from normal prostate (P < 0.001) by Student’s t test.

 


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  		Figure 2. Localization of AT1 receptors in the periurethral region of human prostate by emulsion autoradiography and immunohistochemistry. Photomicrographs (dark-field) of AT1 receptor binding (silver grains) in normal prostate (A) and BPH (B; magnification, x100). Immunostaining of AT1 receptors in normal prostate (C and G) and in BPH (D and H) and corresponding (normal nonimmune serum) negative controls (E and F). Magnification: C–F, x100; G and H, x200. High levels of AT1 receptor binding and staining were localized to periurethral stromal smooth muscle in the normal human prostate and appeared to be reduced in BPH.

 
Immunohistochemistry
Figure 2Go (C–H) shows the results of the immunohistochemical studies using the specific AT1 receptor monoclonal antibody 6313/G2. In the normal prostate, strong intensity of AT1 receptor staining was observed in the periurethral region and was localized to the stromal smooth muscle cells, which appeared to be reduced in BPH (Fig. 2Go, C and G; cf. Fig. 2Go, D and H), confirming the results observed with in vitro and emulsion autoradiography. Background staining (negative control) for the AT1 receptor antibody, as determined by substitution of the primary antibody with normal nonimmune serum (or PBS), was undetectable, indicating the specificity of the antibody used (Fig. 2Go, E and F). AT1 receptors colocalized with {alpha}-smooth muscle actin antibody staining (data not shown).

Ang II receptor binding in human prostatic membranes
Saturation analysis. Specific binding of [125I]-[Sar1,Ile8]Ang II to normal prostatic membranes was saturable, and nonspecific binding ranged from 10–25% (Fig. 4AGo). Scatchard analysis of the data revealed linearity, consistent with the presence of a single class of high affinity binding sites (Fig. 5BGo). There was a significant decrease (~54%) in the maximum number of binding sites (Bmax) in BPH compared with the normal prostate with no significant change in the apparent dissociation constant (Kd) or Hill coefficient values (Fig. 5Go, A and B, and Table 1Go). Prostatic membranes were also prepared from the lateral region of the human prostate, but little or no specific [125I]-[Sar Sar1,Ile8]Ang II binding was observed (data not shown), confirming the in vitro autoradiographic findings.



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  		Figure 4. A, Saturation binding of [125I]-[Sar1,Ile8]Ang II to normal human prostatic membranes ({circ}, total binding; •, specific binding; {lozenge}, nonspecific binding). Nonspecific binding was determined in the presence of 10 µM Ang II amide. The data shown represent the mean of five determinants performed in duplicate. B, Displacement of specific [125I]-[Sar1,Ile8]Ang II binding to normal human prostatic membranes by losartan (•) and PD123319 ({circ}). The data shown represent the mean of three determinants performed in duplicate.

 


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  		Figure 5. A, Saturation analysis of specific [125I]-[Sar1,Ile8]Ang II binding to normal prostate ({circ}) and BPH (•) membranes. Specific [125I]-[Sar1,Ile8]Ang II binding was determined as the difference between total and nonspecific binding in the absence and presence of 10 µM Ang II amide. The data shown represent the mean of three to five determinants performed in duplicate. B, Scatchard plots derived from the specific [125I]-[Sar1,Ile8]Ang II saturation binding data for normal prostate ({circ}) and BPH (•).

 

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  		Table 1. Values for the Kd, Bmax, and Hill coefficient of [125I]-[Sar1,Ile8]Ang II binding to normal prostate and BPH

 
Competition analysis. To identify the subtypes of Ang II receptors in both normal prostate and BPH, competitive binding assays were carried out using [125I]-[Sar1,Ile8]Ang II (1 nM) and unlabeled losartan and PD123319 in increasing concentrations (1 x 10-11 to 1 x 10-5 M). Losartan displaced [125I]-[Sar1,Ile8]Ang II in normal human prostate membranes, whereas PD123319 (up to 10 µM) had no effect (Fig. 4BGo). As shown in Table 2Go, the inhibitory concentration (IC50) and the inhibitory constant (Ki) values for losartan and PD123319 were not significantly different in BPH compared with normal prostate.


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  		Table 2. Inhibition of [125I]-[Sar1,Ile8]Ang II binding to normal prostate and BPH by losartan and PD123319

 
Effects of Ang II on [3H]-noradrenaline release
Due to the unavailability of fresh and functionally viable normal human prostatic tissues, functional experiments could only be performed on fresh prostates taken from Sprague Dawley rats. The mean absolute [3H]-noradrenaline release from rat prostates during the first period of stimulation was 3063 ± 562 dpm (n = 5). The mean [3H]-noradrenaline release during the second period of stimulation, expressed as a percentage of that during the first period, was 85.42 ± 3.18% (n = 5). This technique for the detection of [3H]-noradrenaline from sympathetic nerves of the rat prostate has been validated in our laboratory and shown to be due to the neuronal exocytotic release of transmitter noradrenaline. The neuronal Na+ channel blocker tetrodotoxin (1 µM), the neuronal Ca2+ channel blocker {omega}-conotoxin (0.1 µM), or the removal of extracellular Ca2+ from the PSS, each abolished the radioactive efflux evoked by field stimulation (data not shown). The effects of Ang II on S-I [3H]-noradrenaline release from the rat prostate, expressed as a percentage of the control, are shown in Fig. 6Go. Ang II (0.01–1 µM) significantly enhanced S-I [3H]-noradrenaline release in a concentration-dependent manner, with a maximal effect at 0.1 µM of approximately 40% (P < 0.001).



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  		Figure 6. Effects of Ang II on [3H]-noradrenaline release from rat prostates previously incubated with [3H]noradrenaline to radiolabel the noradrenergic transmitter stores. The intrinsic sympathetic nerves of the rat prostate were subjected to two 60-sec periods of electrical field stimulation to evoke the release of radiolabeled transmitter noradrenaline. Where indicated ({blacksquare}), Ang II (0.01–1 µM) was added to the PSS superfusing the prostate preparations 15 min before the second period of stimulation. Data are expressed as a percentage of the control (absence of Ang II). Each columnrepresents the mean ± SEM; the number of tissues is indicated at the base of each column. *, Significantly different from control (0; P < 0.001) by ANOVA and Dunnett’s test.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This paper reports the first demonstration of the distribution and binding properties of Ang II receptors in normal and hyperplastic human prostates. In vitro autoradiography revealed that Ang II receptors in the human prostate are predominantly of the AT1 subtype and are densely populated in the periurethral region. Light microscopic autoradiography and immunohistochemistry showed that AT1 receptors were localized to stromal smooth muscle, which was confirmed by staining for {alpha}-smooth muscle actin. In many tissues, Ang II is well known to enhance smooth muscle tone (29, 30), stimulate cellular proliferation and growth (31), and facilitate sympathetic transmission (8, 32, 33). The observation that AT1 receptors are concentrated around the periurethral region of the human prostate suggests that Ang II may be involved in modulating smooth muscle tone and growth and, hence, control of micturition. It is possible therefore that Ang II may promote cellular growth and enhance sympathetic tone in the prostate, two major factors that contribute to the pathophysiology and symptomatology of BPH (3).

The unique pattern of AT1 receptor binding in the human prostate was observed in all normal sections regardless of age, and the number of receptors was markedly reduced in all BPH sections studied. These observations suggest that down-regulation of AT1 receptors is not an age-related phenomenon per se, but, rather, a specific feature of BPH. We have also shown, for the first time, Ang II receptor binding to prostatic membranes derived from normal and BPH human tissues. Ang II binding properties in the human prostate are comparable to those reported previously for other tissues, such as the human heart (34, 35). The binding characteristics of [125I]-[Sar1,Ile8]Ang II to prostatic membranes are highly specific with only one binding site and Hill coefficients close to unity. Saturation binding studies revealed a significant decrease in the binding capacity in BPH compared with that in normal prostate (17.86 ± 1.96 and 38.45 ± 3.76 fmol/mg protein, respectively), suggesting that the AT1 receptor down-regulation seen in BPH with in vitro autoradiography and immunohistochemistry is due to a decrease in receptor numbers. In addition, we demonstrated the predominance of AT1 receptors in normal prostate and BPH, in that the AT1 receptor antagonist losartan (Ki, 11.60 ± 1.64 and 10.87 ± 1.80 nM, respectively) selectively displaced [125I]-[Sar1,Ile8]Ang II-binding sites, whereas the AT2 receptor antagonist PD123319 (>10,000 nM) did not.

There is strong evidence to suggest that enhanced sympathetic activity represents a major feature in the development of BPH and associated urinary obstruction. Indeed, {alpha}1-blockers such as prazosin and doxazosin have proved useful in relieving urinary symptoms in patients with BPH (36, 37). It is well established that Ang II can facilitate sympathetic transmission by enhancing the release of the chemical transmitter noradrenaline from sympathetic nerve terminals (8, 33, 38). The facilitatory effect of Ang II on noradrenaline release has been observed in many cardiovascular tissues, including heart, blood vessels, and kidney (8, 33). The present study has shown for the first time that Ang II can also facilitate noradrenaline release in the rat prostate, suggesting a functional role of Ang II in modulating sympathetic transmission in the prostate. It is possible therefore that overactivity of the RAS may be involved in the pathophysiology of BPH by increasing local sympathetic activity.

The findings of the present study provide strong evidence that AT1 receptors predominate in the human prostate and that AT1 receptors are markedly down-regulated in BPH, as confirmed and reinforced by several different techniques. It is well established that AT1 receptors are subject to regulation both in vivo and in vitro. Activation of the RAS, resulting in increased levels of Ang II, is known to down-regulate or internalize AT1 receptors due to agonist-induced stimulation (39, 40, 41). For example, Nickenig et al. showed that in transgenic (mRen-2) 27 rats, an in vivo animal model with a hyperactive RAS, AT1 receptors were down-regulated in the aorta and heart due to AT1 receptor stimulation by increased local levels of Ang II (40). Conversely, inhibition of the RAS, with subsequent reductions in Ang II levels, caused an up-regulation of AT1 receptors (42). Furthermore, AT1 receptor down-regulation has been demonstrated in other disease states, such as congestive heart failure, in which hyperactivity of the cardiac RAS is implicated. Specifically, AT1 receptors were shown to be significantly reduced and ACE-binding sites were increased in the failing human heart compared with those in the normal heart (43, 44). It is reasonable therefore to suggest that the AT1 receptor down-regulation seen in BPH is due to increased tissue levels of Ang II in the prostate. In support of this contention, we have also shown in other studies that ACE messenger RNA and protein (45 ; and Nassis, L., A. G. Frauman, M. Ohishi, J. Zhou, D. J. Casley, C. I. Johnston, and M. E. Fabiani, manuscript submitted) and Ang II peptide (Dinh, D. T., A. G. Frauman, M. Ohishi, J. Zhou, D. J. Casley, G. R. Somers, C. I. Johnston, and M. E. Fabiani, manuscript submitted) are increased in BPH, suggesting that the local prostatic RAS may be hyperactive in BPH.

In conclusion, Ang II receptors are predominantly of the AT1 receptor subtype in the human prostate, are highly concentrated around the periurethral region, and are localized to stromal smooth muscle. Furthermore, Ang II can facilitate noradrenaline release from the prostate. These findings suggest that Ang II may play a functional role in modulating cellular growth and sympathetic activity in the prostate, and possibly micturition. Moreover, AT1 receptors appear down-regulated in BPH compared with the normal human prostate, which may be due to increased local levels of Ang II in BPH. Taken together, these data support the novel concept that hyperactivity of the local RAS may play a role in the development of BPH.


   Acknowledgments
 
Many thanks to Dr. John Risvanis for helpful discussions and expert advice.


   Footnotes
 
1 This work was supported by grants from the National Health and Medical Research Council of Australia/Commonwealth Department of Veterans Affairs, Sir Edward Dunlop Medical Research Foundation, and the Clive and Vera Ramaciotti Medical Research Foundation. Back

2 Recipient of the Dora Lush Postgraduate Biomedical Research Scholarship from the National Health and Medical Research Council of Australia. Back

Received October 11, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Suzuki H 1998 Treatment of benign prostatic hyperplasia and hypertension in elderly hypertensive patients. Br J Urol [Suppl 1] 81:51–55[CrossRef]
  2. Maruenda J, Bhatnagar V, Lowenthal DT 1999 Hypertension in the elderly with coexisting benign prostatic hyperplasia. Urology [Suppl 3a] 53:7–12
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