Decreased contractile effect of endothelin-1 on hyperplastic prostate

ISSN 0306-3623/96 $15.00 + .00 SSDI 0306-3623(95)00117-4 All rights reserved

Gen. Pharmac. Vol. 27, No. 6, pp. 1061-1065, 1996 Copyright © 1996 Elsevier Science Inc. Printed in the USA. ELSEVIER

Decreased Contractile Effect of Endothelin-1 on Hyperplastic Prostate Nobuo Moriyama, 1, Sigeharu Kurimoto, 1 Noriyuki Miyata, 2 Hiroko Yamaura, 2 Ryuzaburo Yamazaki, 2 Katsumi Sudoh, 3 Osamu Inagaki, 3 Toichi Takenaka 3 and Kazuki Kawabe 1 1DEPARTMENTOF UROLOGY,FACULTYOF MEDICINE, THE UNIVERSITYOF TOKYO, 7-3-1 HONGO, BU~qKYO-KU,TOKYO 113, JAPAN, [TEL: 81-3-3815-5411, EXT. 3566; FAX: 81-3-3816-0554] 2RESEARCHCENTER, TAISHO PHARMACEUTmALCO. LTD., 1-403 YosmNoc8o, OHMIYA, SAITAMA330, JAPAN AND 3INSTITUTE FOR DRUG DISCOVERYRESEARCH, YAMANOUCI-IIPHARMACEUTICALCo., LTD., 21 MIYUKIGAOKA,TSUKUBA,h3ARAKI305, JAPAN

ABSTRACT. 1. The contractile activity, binding activity and localization of endothelin (ET)-I were evaluated in human nonhyperplastic (control) and hyperplastic prostates. 2. ET-1 caused contraction of both prostates in a dose-dependent manner. However, this contraction was markedly decreased in hyperplastic prostates. 3. B~ax and Ka values of hyperplastic prostates were greater than those of the control. 4. The muscle and proliferative epithelium of hyperplastic prostates showed strong staining for the anti-ET-1 antibody. However, the glandular epithelium of control prostates was weakly stained. 5. These findings indicate that responsiveness to ET-1 is decreased, though the ET-1 and ET-1 receptors increase in the hyperplastic prostate. Namely, the increase in ET-1 receptors is not effective in regulating the contractile response of the prostate, because its expression is rather dominant in proliferated gland. 6. These suggest that ET-1 may not have an important role in the release of the obstructive symptoms of benign prostatic hypertrophy. OEN PHARMAC27;6:1061--1065, 1996. KEY WORDS. Prostate (human), benign prostatic hyperplasia, endothelin-1 INTRODUCTION Endothelin (ET)-I is a potent vasoconstrictor peptide produced mainly by vascular endothelial cells (Yanagisawa et al., 1988). ET1 contracts nonvascular smooth muscles, such as those in the bronchus (Henry et al., 1990), urinary bladder (Maggi et al., 1989), and prostate (Morita et al., 1993; Kondo and Morita, 1993; Langenstoer et al., 1993; Kobayashi et al., 1994a; 1994b). The group of Lepor used prostatic tissue obtained during radical prostatectomy for lowvolume prostate cancer to evaluate the binding property of ET-1 (Langenstroer et al., 1993; Kobayashi et al., 1994a; 1994b). Another group compared the total number of ET-1 receptors in nonhyperplastic and hyperplastic prostates (Morita et al., 1993; Kondo and Morita, 1993). However, the contractile effects of ET-1 and other factors, such as binding properties or localization in nonhyperplastic prostate and in hyperplastic prostates, have not been compared. The purpose of the present investigation was to examine the effect of ET-1 in human benign prostatic hypertrophy, through contractile activity, receptor properties and localization by estimating ET-1-like immunoreactivity in isolated nonhyperplastic and hyperplastic prostates. MATERIALS A N D METHODS T i s s u e Specimens Prostates from 5 men aged 61 to 74 obtained at total cytoprostatectomy for bladder tumor and pathologically confirmed to have no benign prostatic hyperplasia (BPH) or tumor invasion, were used as control specimens. Hyperplastic prostates were obtained from 10 *To whom all correspondence should be addressed. Received 16 March 1995.

men aged 56 to 76 at suprapubic prostatectomy, cx-Adrenoceptor blocking drugs and hormonal drugs had not been used before surgery.

M e a s u r e m e n t of tension Tension was measured in specimens of control prostate from 4 men and hyperplastic prostate from 5 men. Prostatic strips (200-300 mg each, about 3-5 mm in diameter and 15 mm in length) were obtained immediately after surgery in a longitudinal direction (LD) and circumferential direction (CD) relative to the urethra. These were placed in ice-cold oxygenated modified Krebs-Hense[eit solution (KHS: NaC1 118 raM, KC1 4.7 mM, NaHCO~ 25.0 nM, CaC12 1.8 mM, MgSO4 1.2 mM, NaH2PO4 1.2 raM, and dextrose 11.1 mM) before use. Each strip was then suspended in a well-oxygenated bath filled with 10 ml KHS at 37°C with one end connected to a tissue holder and the other to a force-displacement transducer (TB612T; Nihon Kohden, Tokyo, Japan). The tissue was equilibrated for 60-90 rain under a resting tension of 1.0 g. The KHS was replaced every 20 min during the experiment. After equilibration, cumulative concentration response curves to ET-1 (10 -9 to 10 -°) (Peptide Institute, Osaka, Japan) and phenylephrine (10 _7 to 10 -4 M) (Sigma Chemical Co. St. Louis, MO, USA) were obtained. Each prostatic strip was exposed to only one contractile agent. After the experiment, cross-sectional areas (CSA) of the specimens were calculated using tissues embedded in paraffin.

M e m b r a n e preparation a n d binding assay Membranes from specimens of control prostate from 3 men and hyperplastic prostate from 10 men were finely minced with scissors and

1062 homogenized in 50 volumes of ice-cold homogenization buffer (0.25 M sucrose containing 50 mM Tris-HCl and 1 mM MgCI2, pH 7.4) with a Polytron PT-10 three times for 15 seconds each. The homogenate was centrifuged at 500xg for 15 min at 4°C. The supernatant was filtered through a single layer of nylon mesh and centrifuged at 50,000×g for 20 rain at 4°C. The pellet was washed twice with icecold incubation buffer (50 mM Tris-HC1, 10 mM MgCI> pH 7.5) by repeated resuspension and recentrifugation. The final pellet was resuspended in ice-cold incubation buffer, yielding a protein concentration of 1 mg/ml, and stored at -80°C until use. The binding assay was performed as follows: ET-1 receptor density was determined in saturation experiments by incubating membrane aliquots (approximately 20 txg protein) with increasing concentrations of [12sI]ET-1(0.003-0.8 nM) (81.4 Tbq/mmol, DuPontMEN, Boston, MA, USA) in final volume of 0.25 ml for 120 rain at 25°C. Incubations were terminated by rapid filtration through a Whatman GF/C filter using a Brandel cell harvester. The filters were rinsed three times with 3 ml aliquots of ice-cold incubation buffer. Radioactivity retained on the filters was then counted with a gamma counter (ARC-950: Aloka Co Ltd., Tokyo, Japan) with an efficiency of 81.0%. All assays were performed in duplicate. Nonspecific binding was determined in the presence of 0.1 IzM ET-1. The protein content of each membrane suspension was measured by the method of Bradford (1976).

N. Moriyama et al. 800 s00 "~

~ 400 2oo

0 8

7

6

-log (Endothelln-1) M

500 400 -~ $ 300

....••...-- Cont. (LD) +Cont. "-,~-

.~.~..-~

(CD}

BPH(LD)

~

/ ~ < ~ 1 ~

~. aoo 100 0 6

Immunohistochemical staining All prostate tissues were used for immunohistochemical staining. The avidin-biotin complex method was used for immunohistochemical staining. All staining procedures were carried out at room temperature. Five Ixm frozen sections were cut from O.C.T. compound (Tissue-Tek®) tissue blocks and fixed with buffered formalin (15 min). After exposure to a mixture of 0.03% hydrogen peroxide in methanol to inhibit endogenous peroxidase activity, the sections were treated with blocking serum (normal goat serum) to block nonspecific protein binding and incubated for 2 hr in the anti-human ET-1 polyclonal antibody (rabbit antiserum) (1:200 dilution with phosphate buffered saline; PBS, pH 7.4) (Peptide Institute Inc., Osaka, Japan). The avidin-biotin-complex kit (Vector Laboratories Inc., Burlingame, CA USA) was then adapted to the staining method. The specimens were incubated with biotinylated secondary antibody for 30 rain, further with the avidin-biotin complex, and the final complex was visualized using 0.05 % 3-3'-diaminobenzidine and 0.005% hydrogen peroxide in TrisHCl buffer, pH 7.6. The sections were then counter-stained with hematoxylin, dehydrated and mounted.

Data a n a l y s i s Results are expressed as means+SE. Regression lines were calculated by the least squares method. The contractile forces of prostate from control and BPH were expressed as mg tension. Mean effective concentrations (ECs0; concentration of contractile agent which produces 50% of maximal response) were obtained from the log concentration-response curve. Differences between mean values of tension (mg) and gram tension/mm 2 CSA, respectively, were evaluated by the analysis of variance followed by Scheffe,'s multiple comparison method at a level of significance of p<0.05 and p<0.01. ECs0 and maximal contraction were also evaluated by the Mann-Whitney U test. The saturation curves were analyzed using the nonlinear curves fitting program Ligand (Munson et al., 1980) to determine the ap-

5

4

-log (Phenylephrlne) M FIGURE 1. Concentration-response curves of prostatic strips from control (solid symbols) and BPH (open symbols) patients to ET-1 (upper) and phenylephrine (lower). Results are expressed as the developed tension (mg) with means+-SE LD, longitudinal direction; CD, circumferential direction.

parent dissociation constant (Ka) and maximum number of binding sites (Bm~ x) for [t25I] ET-1. Staining of the anti-ET-1 antibody was scored from ( - ; score 0) to (+ + +; score 3) according to an arbitrary modification of staining intensity and incidence evaluation method (Aoki et al., 1989). Average scores of the control and BPH groups were calculated using the Mann-Whitney U test. RESULTS

Contractile response to E T . 1 In control prostates, ET-1 contracted LD strips and CD strips in a similar dose-dependent manner (Fig. 1). The contraction by ET-1 induced desensitization, and the second application of ET-1 failed to contract the prostatic strips. The EC~0 values for ET-1 were 2.97+1.10x10 -~ M (n=4) for LD strips and 3.50+-2.06x10 8 M (n=4) for CD strips. Maximal contraction was 753-+ 124 mg (n =4) for LD strips and 682-+117 mg (n=4) for CD strips. The gForce/ mm 2 CSA was 0.082+_0.004 for LD strips and 0.076+_0.011 for CD strips (Fig. 2). There was no significant difference between ET-l-induced contraction of the LD strips and CD strips in these control prostates. ET-1 also contracted the hyperplastic strips in a dose-dependent manner (Fig. 1). However, the maximal contraction induced by ET1 in these strips was significantly lower than that in controls (p<0.01). ECs0 values for ET-1 were 11.6-+2.80× 10 -s M (n=5) and 9.40 + _ 1.80x 10-8M (n=5), and maximal contraction was 258_+54 mg (n=5) and 268+-75 mg (n=5) in LD and CD strips, and the g

Decreased Effect of Endothelin-1 on Hyperplastic Prostate

ET-1

1063 The mean Hill coefficient was 0.96+_0.03 in the control and 1.02 +_0.01 in hyperplastic prostates, including that [1>I] ET-1 identified a single population of binding sites (Table 1).

0.1 0.08 e~ E 0.06 0.04

Localization of ET-1 in the prostate

0.02

0 Cont. (LD)

BPH'(LD)

Cont. (CD)

BPH (CD)

Phenylephrine

0.1 0.08 0.06

0.04 0.02 0 Cont. (LD)

BPH (LD)

Cont. (CD)

BPH (CD)

FIGURE 2. E ~ values are shown for 10 4 M phenylephrine and 10 6 M ET-1. Em~ value represents mean+_SE and is expressed as gram tension/mm 2 cross-section areas. T h e g Force/mm 2 CSA on ET-1 is significantly lower in hyperplastic prostates ( n = 5 ) than in control prostates ( n = 4 ) . Those on phenylephrine show no significant difference in both groups of prostates.

Force/ram2 CSA was 0.034---0.007 for LD strips and 0.034+-0.007 for CD strips, respectively, from men with BPH (Fig. 2). These values were significantly lower than those for control specimens (p<0.01). There was no significant difference between ET-l-induced contraction of the LD and CD strips in these hyperplastic prostates.

Contractile response to phenylephrine In control prostates, phenylephrine contracted both LD and CD strips in a dose-dependent manner (Fig. 1). ECs0 values for phenylephrine were 8.10+-0.70x 10 6 M (n=4) for LD strips and 5.90+- 1.00 × 10 -6 M (n=4) for CD strips. Maximal contraction was 387+-63 mg (n=4) for LD strips and 325+50 mg (n=4) for CD strips. The gForce/mm2 CSA was 0.049+-0.009 for LD strips and 0.042+-0.005 for CD strips (Fig. 2). In hyperplastic prostates, phenylephrine also contracted the strips in a dose-dependent manner (Fig. 1 ). However, in contrast to contraction with ET-I, phenylephrine-induced contraction was similar to that of controls in both directions. The EC50values for phenylephrine were 5.79+_0.98×10 ~' M (n=5) and 4.25+-0.86×10 6 M (n=5), and maximal contraction was 322+41 mg (n=5) and 318+-76 mg (n=5) for LD and CD strips, respectively. The gForce/ m m 2 CSA was 0.044+-0.003 for LD strips and 0.041+-0.006 for CD strips (Fig. 2). There was no significant difference between phenylephrine-induced contraction in the LD strips and CD strips in hyperplastic prostates. However, the contractions induced by phenylephrine were significantly less than the contraction induced by the ET-1 (p
Binding experiments Ki values of [125I] ET-1 were 0.048+-0.009 nM in the control and 0.178_+0.23 nM in hyperplastic prostates, this difference being significant (p<0.01). The B,,,~ of [125I] ET-1 was 745.7 + 12.4 fmol/mg protein in the control and 1893.9+272.2 fmol/mg protein in the hyperplastic prostates, this difference also being significant (p<0.01).

In control specimens, ET-l-like immunoreactivity was observed in the muscle layers of the interstitium (score: 2.40+0.24) and in the cytoplasm of glandular epithelium (score: 1.40+_0.24). However, the staining of epithelial cells of the gland was weaker than that in the muscle layers (p
1064

N. Moriyama et al. TABLE 1. /Ca, Bm~xand Hill coefficient values of the ET-1 of prostate specimens (control and BPH) Ka (nM)

Bma~ (fmol/mg/protein)

Hill coefficient

Control I 2 3 mean

0.043 0.036 0.065 0.048 ± 0.009

769 727 741 746 ± 12

1.01 0.933 0.932 0.958 ± 0.026

BPH l 2 3 4 5 6 7 8 9 10 mean

0.091 0.153 0.116 0.288 0.236 0.240 0.089 0.132 0.185 0.247 0.178 ± 0.023

2365 864 1512 764 3431 1128 1455 2260 948 2434 1894 ± 272

1.024 1.102 1.029 1.009 0.996 1.006 1.012 1.015 1.001 1.011

p < 0.01

p < 0.01

P value

1.021 ± 0.01

gested that the binding activity of ET-1 receptors was lowered in hyperplastic prostates. However, in the contractile experiment, ECs0 was not changed. This result was hard to interpret and insufficient to explain the decreased contraction to ET-1. As for the intracellular pathway, changes of smooth muscle cells are a problem. Ultrastructurally, the muscle cells in hyperplastic prostates contain more organdies and fibers than those in normal prostate (Bartsch et al., 1979). Thus, the nature of muscle cells in hyperplastic prostates is different from that in normal prostate. Despite the evidence for the alteration of muscle cells, it is difficult to evaluate the precise mechanism of stimulation through ET- 1 receptors. It is still controversial as to whether or not an intracellular pathway in smooth muscle cells effects the difference of the response. The support for this relative decrease comes from the finding of prominent ET-l-like activity and/or receptors in the epithelium of the gland (Kondo and Morita, 1993; Langenstroer et al., 1993; Sakurai et al., 1992). The present results showed that the intensity of ET-1 antibody staining was markedly increased in proliferative epithelial cells of hyperplastic prostates, and considered to correspond to the increase in B,n~xin the hyperplastic prostate. Thus, the decrease in contractivity is considered to be caused by the relatively decreased binding of ET-1 to receptors in muscles compared to that in epithelial glands. In conclusion, ET-1 appears to play a role as a dynamic component in smooth muscle contraction of nonhyperplastic and hyperplastic prostates. In our specimens, however, contraction was significantly weaker in hyperplastic prostates than control prostates, presumably because of an increase in binding of ET-1 to the glandular cells and a relative decrease in binding to the smooth muscle cells. It is, therefore, possible that the blockage of ET-1 receptors may not be in the treatment of the obstructive symptoms of bladder outlet obstruction in BPH. FIGURE 3. Staining against ET-1 is strongly recognized in the cytoplasm of the proliferative epithelial gland. Muscle cells in the stroma also show positive staining (upper). Epithelial cells from control prostate show weak positive staining, despite good staining of the stromal muscle cells (lower).

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1065 Langenstroer P., Tang R., Shapiro E., Divish B., Opgenorth T. and Lepor H. (1993) Endothelin-1 in the human prostate: tissue levels, source of production and isometric tension studies. J. Urol. 150, 495-499. Maggi C. A., Giuliani S., Patacchini R., Santiciotli P., Turini D., Barbanti G. and Meli A. (1989) Potent contractile activity of endothelin on the human isolateral urinary bladder. Br. J. Pharmac. 96, 755-757. Morita T., Ando K., Kihara K., Matsumura T., Kamai T. and Oshima H. (1993) Effects of endothelin- 1 on the smooth muscle contractivity of human urinary bladder, spermatic cord and prostatic adenoma. Nippon Hinyoukika Gakkai Zasshi 84, 1649-1654 (in Japanese). Munson P. J. and Rodbard D. (1980) Ligand: a versatile computerized approach for characterization of ligand-binding systems. Anal. Biochem. 107, 220-239. Sakurai T., Yanagisawa M. and Masaki T. (1992) Molecular characterization of endothelin receptors. TIPS 13, 103-108. Yanagisawa H., Kurihara M., Kimura S., Tomobe Y., Kobayashi M., Mitsui Y., Yazaki Y., Goto K. and Masaki T. (1988) A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature 332, 411-415.