LOCALIZATION AND EXPRESSION OF THE alpha1A-1, alpha1B AND alpha1D-ADRENOCEPTORS IN HYPERPLASTIC AND NON-HYPERPLASTIC HUMAN PROSTATE

0022-5347/99/16 12-0635$0:3.00/0 Tile JOIYISAI. OF UHOLOGY Copyright 0 1999 by A M E H I ~ A UROLOGI~AL N ASSO(UTIOX. INC.

Vol. 161, 635-640, February 1999 Printed i n U.S.A.

LOCALIZATION AND EXPRESSION OF THE (~1A-1, ( Y ~ AND B (Y~DADRENOCEPTORS IN HYPERPLASTIC AND NON-HYPERPLASTIC HUMAN PROSTATE PAUL D. WALDEN,* CARL GERARD1 AND HERBERT LEPOR From the Departments of Urology, Pharmacology and Biochemistry, New York University Medical Center, New York, New York

ABSTRACT

Purpose: To determine the expression and localization of the alBand a,,-adrenoceptor (AR) subtypes in hyperplastic and non-hyperplastic human prostate tissue. Materials and Methods: The expression of the a,-AR subtypes w a s examined at the mRNA level by quantitative solution hybridization, and at the protein level by immunohistochemistry using subtype selective antibodies. Results: While the overall level of a,-AR mRNA w a s not significantly different between hyperplastic and non-hyperplastic tissue, there were significant differences in the ratio of the a,-AR subtypes expressed in the two tissue types. The most significant finding from these studies w a s the reduced expression of the alb-ARmRNA in both glandular and stromal hyperplasia. By immunohistochemistry, the CY,~.~-AR w a s detected in the stroma and not in the glandular epithelium. The alB-ARw a s localized predominantly in the epithelium a n d w a s weakly present in the stroma. Lower levels of the alB-ARwere detected in the hyperplastic prostatic epithelium. The alD-ARw a s detected in areas of stroma and w a s abundantly present in blood vessels. Conclusions: The alA.l-, alB- and aID-AR subtypes are differentially localized in h u m a n prostate, and the expression levels of all three subtypes are altered in BPH. Alterations i n a,-AR subtype expression (particularly the aIB-AR)in BPH cannot be solely attributed to changes in tissue morphometry resulting from hyperplasia and m a y be of significance in the pathogenesis of

BPH. KEYWORDS:prostate, prostatic hyperplasia, adrenoceptor, G-protein coupled receptor

The human prostate receives a rich nerve supply from both sympathetic and parasympathetic divisions of the autonomic nervous system.' Cholinergic parasympathetic stimulation increases secretory activity of the prostate, whereas sympathetic stimulation increases smooth muscle tone resulting in the expulsion of prostate secretions into the urethra.' Sympathetic nerves produce the catecholamine neurotransmitter norepinephrine (NEL3 Large amounts of NE are present in the adult human prostate4 consistent with abundant sympathetic innervation. G-protein coupled adrenoceptors ( U s ) mediate cellular responses to NE and it is the a,-ARs that mediate contraction of prostate smooth m ~ s c l e . ~ Not only does sympathetic innervation control prostate smooth muscle tone, but accumulating evidence from animal models indicates that the sympathetic nervous system also has a trophic function in prostate growth and development, supporting the concept of an association between sympathetic activity and BPH.' In the sexually immature rat, unilateral sympathectomy leads to decreased ventral prostate weight and DNA content in the lesioned side.7 Conversely, hyperplasia of the ventral prostate is seen in the spontaneously hypertensive rat (SHR),8 an animal model known to have increased sympathetic activity and increased serum levels of cate~holamines.~ Furthermore, sympathomimetic stimulation of rats by subcutaneous administration of catecholamines produces epithelial hyperplasia of the ventral prostate." Interestingly, men receiving the a,-AR antagonist doxazosin as medical therapy for BPH had significantly increased mean apoptotic indices in both the glandular epi-

thelial(6%) and smooth muscle cells (15%) after 3 months of treatment. Gene cloning studies initially identified three subtypes of the a,-AR, alMa-,alBh- and a I D l d -with , uppercase letters denoting the native pharmacologically defined subtypes and lowercase letters denoting the corresponding molecular cloned subtypes.12 More recently 4 isoforms of the a,,-AR have been identified,13.14generated by alternate splicing of a single gene. These 4 isoforms have been denoted alNa.,-, All three a,-AR subtype mRNAs p - , a1Ma.3and are present in the human p r ~ s t a t e . 'By ~ in situ hybridization and receptor autoradiography using subtype selective limRNNprotein was the gands, it was shown that the a,,,-AR most abundant subtype expressed and was predominantly localized in the stromal compartment of the human prostate.15-17 Due to differences in protein stability andor translation, mRNA expression patterns may not give an accurate picture of protein expression. Furthermore, receptor autoradiography does not offer the necessary degree of resolution to determine receptor expression at the cellular or subcellular level. Recently, a,-AR subtype selective antisera have become available from Santa Cruz Biotechnology that offer the necessary degree of sensitivity to definitively localize a,-AR expression at the cellular and subcellular level in the prostate. The objective of this study was to utilize these newly available molecular tools to provide a side-by-side comparison of a,-AR subtype expression in hyperplastic and nonhyperplastic human prostate tissue.

''

MATERIALS AND METHODS

Accepted for publication August 13, 1998. Materials. Goat antibodies raised to synthetic peptide fiag* Requests for reprints: Department of Urology, Skirball lOU, 550 ments corresponding to the carboxy-termini of the human First Avenue, New York, NY 10016. alB-and a I D -ARs were obtained from Santa Cruz Supported in part by NIH grants DK46416 and CA72290. 635

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a,-ADRENOCEPTORS IN THE PROSTATE

Biotechnology (Santa Cruz, CAI. No cross-reaction was observed between antibodies as assessed using Rat-1 fibroblasts expressing the individual alA.l-r alB-and aID-A R s . I 8 Human a,,.,-, a,,,- and a , d- AR cDNA clones were a kind giR of Pfizer Central Research (Pfizer, Sandwich, Kent, UK). Immunohistochemistry supplies were obtained from Dako (Carpinteria, CA) or Biomeda (Foster City, CA). Tissue procurement. The Institutional Board of Research Associates at NYU Medical Center approved all procedures involving human tissue. Human prostate tissue was obtained from male patients undergoing suprapubic prostatectomy for BPH or retropubic prostatectomy for low-volume organconfined prostate cancer. Only transition zone prostate tissue was used in this study. Prostate tissue used in this study was obtained from patients with a mean age of 60.6 years (range, 49 to 66 years), a mean PSA level of 7.4 (range, 1.1 to 21 ng./ml.) and with Gleason scores of 5 6. Transition zone tissue was dissected away from the gland and was cut into cubes < 10 mm. in dimensions, snap frozen in liquid nitrogen and stored at -8OC. Tissue specimens were sectioned in a cryostat (Jung CM3000) and subjected to histological analysis and morphometry prior to RNA isolation according to the following procedure. Prior to chilling to -2OC, the inside of the cryostat was rinsed with a 2% solution of Absolve (NEN, Boston, MA) to remove contaminating RNase. The frozen tissue was placed inside the cooled cryostat, allowed to equilibrate to the temperature of the chamber and embedded in Tissue-Tek OCT 4583 (Miles, Elkhart, IN). The tissue was cut into 500 pm. slices and collected in a RNase free tube pending analysis of tissue histology and morphometry. Random 5 pm. sections were also cut throughout the tissue block and adhered to positively charged glass microscope slides (Fisherbrand Superfrost plus, Fisher Scientific, Springfield, NJ) for analysis of histology (by experienced uro-pathologist Jonathan Melamed in the Department of Pathology, NYU Medical Center) and m~rphometry.'~ If the histology and morphometry were consistent throughout the tissue block, the sections in the tube were used for RNA isolation. For immunohistochemistry, 5 pm. sections of transition zone prostate tissue were cut and adhered to positively charged glass microscope slides. Non-hyperplastic tissue was obtained from the transition zone of radical retropubic prostatectomy specimens with low-volume cancer confined to the peripheral zone. Although it is rare to find non-hyperplastic tissue in such specimens, over 300 prostatectomies are performed annually a t NYU Medical and associated hospitals. Tissue sampling in this way ensured that our hyperplastic and non-hyperplastic tissues were age-matched, and that any differences noted could not be attributed to age.

A- 1

RNA isolation and quantitative solution hybridization. RNA was isolated by the method of Chomczynski and Sacchi." Quantitative solution hybridization was performed essentially as described. 15." The probes used for solution hybridization encoded unique C-terminal sequences of the alA.l-,alB-and alD-ARs represented diagrammatically in fig. 1.No cross-hybndization was observed between probes as assessed using RNA isolated from Rat-1 fibroblasts expressing the individual alA.l-,alB-and alD-US.'" Statistical tests. Data from non-hyperplastic and hyperplastic tissue groups were compared by unpaired t tests using the Prism 2.01 software package (Graphpad Inc., San Diego, CA). Zmmunohistochemistry. All steps were performed at room temperature unless otherwise indicated. Sections were fixed in acetone (1minute), dried in a desiccator overnight and hydrated in phosphate buffered saline (PBS) (three 5 minute washes). Nonspecific avidin and biotin sites were blocked by sequentially incubating with avidin (Dako X0590) for 10 minutes, washing with PBS, incubating with biotin (Dako X0590) for 10 minutes and washing with PBS. The sections were incubated with tissue conditioner (Biomeda M37) for 3 minutes and then primary antibody (diluted 1:20 in Tissue Conditioner) was applied overnight at 4C. The sections were washed in PBS and incubated with a 1:400 dilution of a biotin-conjugated rabbit anti-goat secondary antibody (Dako E0466) for 30 minutes. The sections were washed with PBS and streptavidin-conjugated horseradish peroxidase (HRP)(Biomeda, Lab Probe) was applied for 30 minutes. Following a PBS wash, sections were briefly rinsed with water and bound antibodies were localized using 3-amino9-ethylcarbazole as chromogen (Biomeda). Color development was followed by microscopy from 4 to 15 minutes. The color reaction was stopped by rinsing in water and the sections were counterstained with 0.1% Mayer's hematoxylin solution (Sigma Chemical Co., St. Louis, MO) for 3 minutes. Finally the sections were rinsed under running water for 15 minutes and mounted in crystal mount (Biomeda M02). RESULTS

Differential expression of the aIa.,-,alb-and aid- AR mRNAs in hyperplastic and non-hyperplastic human prostate. Messenger RNAs encoding the mlA.,-, alB-and a,,-AR subtypes were quantified in human prostate transition zone tissue without histological evidence of hyperplasia and in tissue with histological stromal or glandular hyperplasia by solution hybridization. While the overall level of al-AR mRNA was not significantly different between non-hyperplastic and hyperplastic tissue types, the results (table), indicate significant differ-

B

D

FIG. 1. Derivation of a,-AFt subtype mRNA specific probes. Putative structure of 7 transmembrane domain G protein coupled a,-AR

subtypes (aiA.l,af and,a,q), with individual amino acids represented by circles. Filled circles represent amino acids coded in RNA probes used or solution ybndization experiments.

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a,-ADRENOCEPTORS I N T H E PROSTATE

TABLE Subtype m~~~ Tissue Histology

of total)

% Stroma a1,

(Ilb

ald

56 2 6.0 66 2 5.2 Non-hyperplastic ( n = 7) 5.2 z 1.3 29 t 2.4 Stromal hyperplasia ( n = 7) 78 2 7.4 85 2 5.9t 0.51 Z 0.26t 15 ? 1.6t Epithelial hyperplasia (n = 7) 39 2 8.1 75 2 4.8 1.9 i 0.75t 23 2 2.3t t p <0.05 comparing t o non-hyperplastic.

ences in the ratio of the a,-AR subtypes expressed in stromal or epithelial hyperplasia compared with non-hyperplastic tissue. Specifically in stromal hyperplasia tissue there was increased expression of ala.,-RmRNA (p =0.033) and reduced expression of the a,,- (p =0.0041) and a l d - R (p =0.0004)-As compared with non-hyperplastic tissue. In glandular hyperplasia tissue there was a significant reduction in the expression of the a,,- (p =0.048)and a,,-AR (p =0.015) &As compared with non-hyperplastic tissue. These results represent the first side-by-side comparison of a,-AR mRNA expression in nonhyperplastic tissue and in tissue with stromal or glandular hyperplasia. Our iindings are consistent with previous quantitative studies of a,-AR mRNA expression in 'normal' prostate tissue in radical prostatectomy specimens15and 'hypertrophied' prostate tissue obtained from open suprapubic prostatectomy specimens.22 Based on the amount of stroma in our tissue samples, alterations in a,,-AR mRNA subtype expression may be a reflection of differences in tissue morphometry. The alterations in expression of the alb- and ald-AR &As, however, were greater than could be explained simply by differences in tissue morphometry. Furthermore, the a,,-AR mRNA was decreased in both glandular and stromal hyperplasia. Localization of the alb-and ald-ARmR.NAs in human prostate by in situ hybridization using radioactively labeled probes could not be discerned above ba~kground.'~. l6 By in situ hybridization using

A.

294 I W.. .MPI

1 I

non-radioactive probes Moriyama et a1 22 detected diffuse expression of the ald-ARmRNA and very low levels of the alb-AR mRNA in interstitial smooth muscle areas. These studies further highlight the need to examine receptor expression at the cellular and protein levels. ,Tocalization of Q I B - and a,D-ms in human prostate. Goat antibodies raised to carboxy-terminal synthetic peptide f r a m e n t s of the human ala.,-, and aln-. ARs were obtainea from Santa Cruz Biotechnology. No crossreaction was observed between antibodies as assessed using Rat-1 fibroblasts expressing the individual alA.,-,alB-and ~,,-ARS.'~ Because the antisera were raised against C-terminal epitopes of the corresponding a,-ARs, the "alA" specific antibody would be expected to recognize only the alA., splice variant (fig. 2). The three antibodies were used to localize the C I , ~alB.~, and a,,-& in frozen sections of human transition zone prostate tissue (fig. 3). The a,*., AR was localized in the stromal compartment and was not detected in epithelial cells (fig. 3, B ) . The alB-ARwas localized predominantly in nonhyperplastic epithelial cells and at very low levels throughout the stroma (fig. 3, C).Expression of the alB-ARwas iower in epithelial cells in areas of glandular hyperplasia (inset to fig. 3, C). The alD-ARwas detected at low levels in areas of stromal tissue that appeared to contain a predominance of smooth muscle cells and was abundant in blood vessels (fig. 3, D).In addition the a,,-AR was detected covering about approximately 50% of a subset of glandular cells (fig. 3, E ) . These glandular cells appeared to be located in areas of ductal branching. Subjectively, using these antibodies we were unable to discern differences in the expression of the stromal alA.,- and a,,-ARs comparing non-hyperplastic to hyperplastic tissue, even though we had detected differences in the expression of the corresponding mRNAs. Our localiza-

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423 I I GSF.. .TAU 295

B. 424

466

FIG. 2. Carbox 1 terminal sequences of four human aIA-AR splice variants. A, organization of gene encoding human a,-AR. Common sequences are in&ated by gray rectan les, with roman numerals referring to seven transmembrane domains. Intruns are shown by thin black lines with exons uni ue to 4 &,a is~forms'~''~ shown as black rectangles. B, representation of four a AR isoforms (ar.,-, aU.*a ~ .and ~ aU. ) generatediy alternate splicing together with deduced carboxyl-terminal amino acid sequences.%trons removed y sphcmg are shown by dotted lines. Sequence of peptide used to generate specific antibodies by Santa Cruz Bioteehology is shown boxed.

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a,-ADRENOCEPTORS IN THE PROSTATE

non-hyperplastic glands. The alB-ARis also poorly expressed in atrophic glands (data not shown). Taken together, the finding of reduced expression of the alB-ARin both stromal and epithelial hyperplasia suggests that reduced expression of the a,,-AR is diagnostic for BPH in general. DISCUSSION

639

ferentiation in the prostate. These effects could be compounded by changes in a,-AR subtype expression observed in hyperplastic compared with non-hyperplastic prostate. It is interesting in this regard that other investigators have shown decreased expression of the a,,,- and ald-AR mRNAs and increased expression of the ala-AR mRNA in cardiac myocytes chronically exposed to NE.25 The involvement of the sympathetic nervous system and the a,-ARs in the pathophysiology of human BPH needs to be addressed. Finally, in light of the demonstration that the non-selective a,-blocker doxazosin causes apoptosis in stromal and epithelial elements of BPH," our results indicate that selective alBblockers and selective a,,-blockers may be useful in the reduction of glandular and stromal components, respectively, of BPH. ~ ~Acknowledgments. .~~ The authors wish to express their gratitude to Drs. Jonathan Melamed (Pathologist, NYU Medical Center) and Rosemary Wieczorek (Pathologist, New York VA Medical Center) for histological analysis of human prostate tissue.

In this study, we performed a side-by-side quantitative analysis of a,-AR mRNA expression and a qualitative examination of a,-AR receptor localization in human prostate tissue with and without histological evidence of hyperplasia. Consistent with previous receptor binding studies,23 we did not detect a significant change in the overall level of a,-AR mRNA comparing hyperplastic to non-hyperplastic prostate tissue. Also, consistent with previous separate s t ~ d i e s , we did detect significant changes in the ratio of the individual a,-AR subtypes comparing hyperplastic to nonhyperplastic prostate tissue. The most significant finding of our studies was the reduced expression of the alb-ARmRNA in both stromal and epithelial hyperplasia compared with non-hyperplastic prostate tissue. A full understanding of the adrenergic physiology of the REFERENCES prostate is dependent upon precise knowledge of the local1. Walsh, P. C. and Donker, P. 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