Alpha1 Adrenoceptor Subtypes in the Human Prostate

0022-534 7/93/1493-0640$03.00/0 Vol. 149, 640-642, March 1993

THE JOURNAL OF UROLOGY Copyright© 1993 by AMERICAN UROLOGICAL ASSOCIATION, INC.

Printed in U.S.A.

ALPHA 1 ADRENOCEPTOR SUBTYPES IN THE HUMAN PROSTATE HERBERT LEPOR,* RUI TANG, SHIMON MERETYK,

AND

ELLEN SHAPIRO

From the Departments of Urology, Pharmacology and Toxicology, Medical College of Wisconsin, Milwaukee, Wisconsin

ABSTRACT

High affinity a 1 adrenoceptors have been characterized in the human prostate. The tension of prostatic smooth muscle is mediated by the a 1 adrenoceptor. The present study represents the first characterization of human a 1 adrenoceptor subtypes using radioligand receptor binding techniques. Binding studies were performed on tissue homogenates obtained from the human prostate. Competitive inhibition studies were performed in the presence of an 80 pl\lI. 125I-Heat and 16 concentrations of unlabelled 5-methylurapidil (5 MU) or WB-4101 (10- 10 M. to 10-5 M.). Saturation experiments were also performed with and without chloroethylclonidine (CEC, 10-5 M.), a compound that selectively inactivates the arn subtype. The individual displacement plots for WB-4101 and 5MU in the human prostate were consistently best fit by a 2 binding site model. WB-4101 and 5MU exhibited a 594- and 186-fold higher affinity for the prostatic a 1A binding site relative to the arn binding site. The ratios ofprostatic a 1A/arn binding sites discriminated by WB-4101 and 5-MU were 1.8 and 1.6, respectively. CEC inactivated 44% of the prostatic a 1 binding sites. The binding studies suggest that the dominant a 1 subtype in the human prostate is the a 1A. We are characterizing the functional properties of the a 1 subtypes in the human prostate. KEY WORDS: prostate; receptors, adrenergic

High affinity a 1 adrenoceptors have been characterized in the human prostate using radioligand receptor binding methods.1· 2 The tension of prostatic smooth muscle is mediated by the a 1 adrenoceptor. 3 • 4 Minneman et al. recently reported that the alpha antagonists WB4101 and 5-methyl urapidil (5-MU) discriminate 2 different alpha1 adrenoceptor binding sites (a1A and arn) using competitive inhibition studies. WB4104 and 5MU were shown to exhibit a higher affinity for the a 1Asubtype. 5 The a 1 subtypes were further characterized by selectively inactivating the arn subtype with chloroethylclonidine (CEC). 6 The proportion of a 1A and arn binding sites varies in different central and.. peripheral tissues. 5 For example, the rat spleen contains primarily the arn subtype, and the rat hippocampus contains primarily the a1A subtype. The a1 adrenoceptor subtypes have not been previously characterized in the prostate. The objective of the present study was to characterize and determine the relative proportion of the a1A and the arn adrenoceptor subtypes in the human prostate. Characterizing the a 1 adrenoceptor subtype that mediates prostatic smooth muscle tension may have important clinical implications related to the pharmacotherapy of benign prostatic hyperplasia (BPH). MATERIALS AND METHODS

Human prostatic adenomas were obtained at the time of surgery from 12 men, aged 55 to 75 years, undergoing open prostatectomy for symptomatic BPH. The weight of the enucleated adenomas ranged between 50 and 150 grams. The tissue specimens for receptor binding assays were immediately transferred into a -70C freezer for storage. Preparation of Tissue Homogenates. The prostate specimens were weighed, immersed directly into liquid nitrogen, and pulverized using a Thermovac tissue pulverizer. The tissue fragments were homogenized in 20 volumes (vol./wt.) of ice-cold 50 mM. potassium phosphate buffer (pH 7.4) with a Brinkmann polytron at a speed setting of 6 for 60 seconds. The homogenates were centrifuged at 40,000 g. in a Sorvall RC-5B centrifuge using a Sorvall SA-600 rotor for 10 minutes at 4C. The tissue preparation for the saturation assays without pretreatment Accepted for publication October, 2, 1992.

* Requests for reprints: Department of Urology, Medical College of Wisconsin, 9200 West Wisconsin Ave., Milwaukee, Wisconsin 53226. This study was supported by NIH Grant IR29-DK43149-0l.

with CEC and the competitive binding assays proceeded by homogenizing the pellets in 20 volumes of Kreb's-Ringer'sPhosphate buffer (pH 7.4) containing 10 µM. NaGTP. The tissue preparation for the saturation assays pretreated with CEC proceeded by homogenizing the pellets in 20 volumes of 10 µM. CEC in 10 mM. NaHEPES buffer (pH 7.4). The homogenates were shaken on a New Brunswick G2 gyrotory shaker at room temperature for 30 minutes at a speed setting of 100 rpm and then centrifuged at 40,000 g. for 10 minutes at 4C. The pellets were washed twice with 20 volumes of ice-cold 50 mM. sodium potassium phosphate buffer (pH 7.4). The pellets were homogenized and centrifuged at 40,000 g. for 10 minutes at 4C between washes. The final pellets were homogenized in 50 volumes of 50 mM. sodium potassium phosphate buffer (pH 7.4) at room temperature and filtered through fine Nitex mesh gauze. Saturation Binding Analysis. Saturation analyses were performed at 7 different concentrations (0.01 nM. to 0.40 nM.) of [125I]-Heat (2-(-( 4-hydroxyphenyl)-ethyl-aminomethyl ]tetralone) (New England Nuclear, Boston, Massachusetts) at constant specific activity (2200 Ci./mM.). Total binding was determined in 500 µl. containing 400 µl. tissue homogenate with and without CEC (Research Biochemical Corp., Natick, Massachusetts) pretreatment, 50 µl. of 50 mM. sodium potassium phosphate buffer (pH 7.4), and 50 µl. of (1251]-Heat at varying concentrations. Nonspecific binding was determined in 500 µl. containing 400 µl. tissue homogenate with and without CEC pretreatment, 50 µl. of 100 µM. phentolamine and 50 µl. of (1251]-Heat at varying concentrations. Total and nonspecific binding determinations for each (1251]-Heat concentration were performed in triplicate in polypropylene test tubes. The assay tubes were shaken at 100 rpm for 30 minutes at room temperature. Maximum specific binding was consistently observed in prostate homogenates following an incubation interval of 30 minutes. The binding assays were terminated by filtering over glass fiber filter paper (Schleicher and Schuell glass fiber no.32) under vacuum on a 24 well Brandel M24R cell harvester (Brandel, Inc., Gaithersburg, Maryland). The glass fiber filters were washed 4 times with 4 ml. of ice-cold 50 mM. sodium potassium phosphate buffer (pH 7.4) containing 10% wt./vol. polyethylene glycol (mol. wt. approximately 3350). The radioactivity was measured on an Automatic Gamma

640

641

ALPHA-1 ADRENGCEPTOR SUBTYPES Il\! PROSTATE

Counter with PC , •. ,.w,,~,,,v-·~ Systems), Scatchard plots, Bmax, and the Hill coefficient of the observed Scatchard plots were generated by the Ligand computer program. 7 Competitive Binding Analysis. Competitive binding experiments were carried out on tissue homogenates obtained from human prostate in the presence of a constant concentration of [125I]-Heat (80 pM.) and 16 concentrations of unlabelled 5MU or WB-4101 (10- 10 M. to 10-5 ). Tissue homogenates were prepared in the manner previously outlined: 400 µl. of tissue homogenates, 50 µl. of 80 pM. [125I]-Heat, and 50 µl. of varying concentrations of 5-MU or WB-4101 were combined and shaken at 100 rpm for 30 minutes at room temperature. The assays were repeated in quadruplicate for each concentration of inhibitors. The assays were terminated as previously described. Nonspecific binding represented [125I]-Heat bound in the presence of 10 µM. phentolamine. Specific binding was determined by subtracting the nonspecific binding component from the total [125I]-Heat binding at the various concentrations of inhibitors. Maximum binding was determined in the presence of [125I]-Heat only. The best two-site fit for a binding curve was calculated by minimizing the sum of the squares of the errors using nonlinear regression analysis. 7 Two-site models were compared to one-site models using a partial F-test to determine whether inclusion of additional parameters in a model significantly increases the "goodness of fit" for the model, more than would be expected on the basis of chance alone. P-values less than 0.05 were considered significant, and then the more complicated model (additional parameters) was the better choice. Statistical Analysis. The 2 sample t-test was used to compare the differences between the Kct and Bmax in saturation studies performed with and without CEC. Competitive displacement experiments were performed on human prostatic tissue homogenates using varying concentrations of unlabelled WB-4101 or 5-MU (10- 10 to 10-s M.) and 125 I-Heat (80 pM.). The composite competitive displacement plots for the 12 WB-4101 displacement assays and 8 5-MU displacement assays are shown in figure 1. The individual displacement plots for WB-4101 and 5-MU in human prostate were consistently best fit by a 2 binding site model. The equilibrium dissociation constants (Kct) and the density of binding sites (Bmax) for the high (H) and low (L) affinity binding sites are presented in table 1. WB-4101 and 5-MU exhibited a higher affinity for the alA subtype relative to the arn subtype. 4•8

Human Prostate 100

o 5-MU 6 WB4101

90 C:

'U

80 70

C:

ffi

60

0 t;::

50

(l)

30

·o 0. (/)

~

40

20 10 0 -10 1E-11

1E-10

1E-9

1E-8

Inhibitor

1E-7

1E-6

1E-5

1E-4

Log[Antagonist], (M) FIG. 1. Competitive displacement experiments were performed on human prostatic tissue homogenates using varying concentrations of unlabelled WB-4101 or 5-MU and 125!-Heat. Composite competitive displacement plots for inhibition of 125 I-Heat by WB-4101 and 5-MU in human prostate are shown.

Kd (nM.)

# Assays

WB4101 5-MU

12 8

Bm'" (fmol./mg. wet wt.)

(H)

(L)

(H)

(L)

0.37 ± 0.06 1.73 ± 0.44

220 ± 41 322 ± 88

0.42 ± 0.04 0.36 ± 0.06

0.24 ± 0.02 0.22 ± 0.02

Representative Scatchard Plot

Human Prostate 0.07 @I - ~ Ii}

without CEC

Kd - 71.74 pM

0.06

Bmax Hill Co.

(!.)

0.05

11.l LL

0.04

= 0.30 fmol/mg = 1.00

wet

wt

L

""'--. 'U C :J 0

m

0()

0.03 0.02 0 - 0

with 1 0 uM CEC

Kd - 77.67 pM Bmax = 0.18 fmol/mg wet wt

0.01

Hill Co.

0.00

=

0

0.97

-t------,-------.---~-.-----~----'0

[1251]-HEAT Bound, pMol FIG. 2. Representative Scatchard plot with and without preincubation with CEC is shown for human prostate. TABLE 2.

RESULTS

cr,

L Displacement of 125!-Heat binding in human prostate by WB-4101 and 5-MU

TABLE

Selective inactivation of human prostatic alpha 18 binding sites by CEC

Saturation Experiments +CEC -CEC P value

Assays

(nM.)

Bma, fmol./mg. wet wt.

5

0.07 ± 0.02 0.11 ± 0.10 0.42

0.18 ± 0.01 0.32 ± 0.01 0.001

#

5

Kd

The Hand L affinity WB-4101 and 5-MU binding sites correspond to the a,A and arn subtypes, respectively. WB-4101 and 5-MU exhibited a 594- and 186-fold higher affinity for the prostatic alA binding site relative to the a 18 binding site, respectively. The ratios of prostatic a,A/ a 18 binding sites discriminated by WB-4101 and 5-MU were 1.8 and 1.6, respectively. The competitive displacement studies indicate that the dominant a, subtype in the human prostate is the a,A. A total of 5 parallel saturation experiments was performed using 125 I-Heat with and without (±) preincubation with 10 mM, CEC. CEC selectively inactivates the a 18 subtype. 5 A representative Scatchard plot with and without preincubation with CEC in human prostate is presented (fig. 2). The binding of 125 I-Heat was consistently saturable and of high affinity in prostate homogenates. The mean Kct and Bmax values determined from the saturation experiments ± CEC in human prostate are summarized in table 2. The a 1 adrenoceptor density in prostatic homogenates was reduced 44 % following CEC pretreatment, suggesting that the ratio of a 1A/ a 18 binding sites is 56:44 (1.3). DISCUSSION

WB-4101 and 5-MU have been shown to discriminate the a,A and arn subtypes in various peripheral and central tissues. 5 In the present study, WB-4101 and 5-MU also discriminated the a,A and arn adrenoceptor binding sites in the human prostate. The relative proportion of a,A and a 18 binding sites in the human prostate determined by competitive displacement stud-

642

LEPOR AND ASSOCIATES

ies with WB-4101 and 5-MU was 1.8 and 1.6, respectively. Minneman et al. have reported that preincubation with CEC selectively inactivates the am binding site. 5 The present study demonstrated that CEC inactivated 44 % of the prostatic a 1 adrenoceptors. The ratio of a1A and am binding sites based upon the saturation studies ± CEC was 1.3. The competitive displacement studies with WB-4101 and 5-MU and the saturation studies ± preincubation with CEC demonstrated that the a 1A binding site is the dominant alpha1 adrenoceptor subtype in the human prostate. The slight discrepancy between the ratios obtained by competitive displacement and saturation studies ± CEC presumably reflects the different methodologies. The findings of the present study may have important clinical implications related to the medical treatment ofBPH. Approximately 70% of males with symptomatic BPH achieve a favorable clinical response following treatment with alpha blockers. 9 The efficacy and toxicity of selective alpha1 blockers in men with symptomatic BPH is dose dependent. 10 The adverse events most commonly associated with selective alpha1 blockers include light headedness, asthenia and dizziness. 10 The development of adverse events in some patients with BPH obscures the potential therapeutic response. The specific a1 subtype that mediates prostatic smooth muscle tension is under investigation in our laboratory. Preliminary isometric tension studies indicate that the tissue tension elicited by phenylephrine is mediated primarily by the am binding sites in the prostate. It is conceivable that the commonly observed adverse events associated with a blockade may be mediated by vascular and cerebral a1A receptors. Therefore, a drug selective for the am subtype may achieve a therapeutic response in BPH with fewer adverse events. Additional studies are necessary to definitely characterize the a1 subtypes mediating prostatic smooth muscle tension and the precise mechanism for the adverse events

associated with selective alpha blockade in BPH. These studies will provide important insights related to developing selective alpha 1 blockers that will achieve maximal efficacy and reduce toxicity in patients with BPH. Acknowledgements. The authors wish to thank Ms. Pamela Cromell for her editorial assistance. REFERENCES 1. Lepor, H. and Shapiro, E.: Characterization of alpha 1 adrenoceptors in human benign prostatic hyperplasia. J. Urol., 132: 1226, 1984. 2. Gup, D. I., Shapiro, E., Baumann, M. and Lepor, H.: Autonomic receptors in human prostate adenomas. J. Urol., 143: 179, 1990. 3. Gup, D. I., Shapiro, E., Baumann, M. and Lepor, H.: The contractile properties of human prostatic adenomas and the development of infravesical obstruction. Prostate 15: 105, 1989. 1. H~,::obln, .T. P., C!lill'\ J.VT. ~ncl rz~1~'7n-ik, E.· Tn

5. 6. 7. 8. 9. 10.

vitro r.h~r::lCtPr-i'Z::lt.inn

of the alpha adrenoceptors in human prostate. Eur. J. Pharmacol., 107: 111, 1985. Minneman, K. P., Han, C. and Abel, P. W.: Comparison of alpha, adrenergic receptor subtypes distinguished by chlorethyclonidine and WB4101. Mol. Pharmacol., 33: 509, 1988. Han, C., Abel, P. W. and Minneman, K. P.: Heterogeneity of alpha 1 adrenergic receptors revealed by chlorethyclonidine. Mol. Pharmacol., 32: 505, 1987. Munson, P. J. and Rodbard, D.: Ligand: a versatile computerized approach for characterization of ligand binding systems. Anal. Biochem., 107: 220, 1980. Gross, G., Hanft, G. and Rugevics, C.: 5-methyl-urapidil discriminates between subtypes of the alphal adrenoceptor. Eur. J. Pharmacol., 151: 333, 1988. Lepor, H., Knapp-Maloney, G. and Sunshine, H.: A dose titration study evaluating terazosin, once-a-day alpha,-blocker for the treatment of symptomatic BPH. J. Urol., 144: 1393, 1990. Lepor, H., Soloway, M., Narayan, P., et al.: A multicenter fixed dose study of the safety and efficacy of terazosin in the treatment of symptoms of BPH. J. Urol., in press.