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Characterization and Localization of Prostatic Alpha1 Adrenoceptors Using Radioligand Receptor Binding on Slide-Mounted Tissue Section
0022-534 7 /93/1506-2002$03.00/0 THE JOURNAL OF UROLOGY Copyright© 1993 by AMERICAN UROLOGICAL ASSOCIATION, INC.
Vol. 150, 2002-2006, December 1993
Printed in U.S.A.
CHARACTERIZATION AND LOCALIZATION OF PROSTATIC ALPHA1 ADRENOCEPTORS USING RADIOLIGAND RECEPTOR BINDING ON SLIDE-MOUNTED TISSUE SECTION SHINYA KOBAYASHI, RUI TANG, ELLEN SHAPIRO
AND
HERBERT LEPOR
From the Department of Urology, Medical College of Wisconsin, Milwaukee, Wisconsin
ABSTRACT
Alpha1 adrenoceptor binding sites have been characterized in prostatic tissue homogenates using radioligand receptor binding studies. The objective of the present study was to characterize and localize prostatic alpha1 adrenoceptor binding sites using slide-mounted tissue sections and the ligand 3 H-prazosin. The present study demonstrated that preincubation is not required; the optimal incubation interval is 40 minutes; and a 1-minute wash (once or twice) maximizes the proportion of specific 3 H-prazosin binding. Saturation studies were performed at 8 different concentrations of 3 H-prazosin ranging between 0.0625 nM. to 8.0 nM. The binding of 3 H-prazosin was consistently saturable and of high affinity. The mean Kd and Bmax determined from 6 saturation studies was 4.16 X 10-10 M. and 1.30 fmol./mg. wet weight, respectively. The pharmacology of these 3 H-prazosin binding sites was characterized using competitive displacement experiments. The mean IC 50 corrected for prazosin, phentolamine and yohimbine was 7.8 X 10- 10 M., 6.0 X 10-9 M. and 2.1 X 10-6 M. The rank order of the IC 50 corrected values indicates that alpha1 binding sites were measured under the assay conditions. In the present study, the mean values for Kd, Bmax and IC 50 corrected are similar to values previously reported using prostatic tissue homogenates. Prostatic tissue sections were apposed to x-ray film after being incubated with 3 nM. 3 H-prazosin (total prazosin binding) and 3 nM. 3 H-prazosin + 8 µM. prazosin (nonspecific prazosin binding). The autoradiograms were analyzed using a computerized analyzing system. The specific radioactive densities of 3 H-prazosin in the stroma and glandular epithelium were 1099 ± 48 pCi/mg. and 163 ± 42 pCi/mg. The present study validates the technique of assaying alpha1 adrenoceptor binding sites on slide-mounted prostatic tissue sections and provides further evidence that alpha1 adrenoceptor binding sites are localized primarily to the stromal elements of the prostate. KEY WORDS: receptors, androgen; prostate
Alpha1 adrenoceptor binding sites have been characterized in prostatic tissue homogenates using radioligand receptor binding methods. 1-6 The amount ofprostatic tissue required for a single saturation assay ranges between 1 to 5 gm. owing to a relatively high proportion of nonspecific binding and low capacity of prostatic alpha1 adrenoceptors. 1-6 Gup et al. reported that 1251-Heat is the preferred ligand for characterizing prostatic alpha1adrenoceptors owing to a lower proportion of nonspecific binding. 3 The cost of the ligand for a single saturation assay using 1251-Heat is approximately 5-fold higher than that of 3 H-prazosin. Young and Kuhar and Kuhar and Unnerstall have described a technique for performing radioligand receptor binding studies on slide-mounted tissue sections. 7 • 8 This technique offers several potential advantages for characterizing prostatic atpha 1 adrenoceptors since less tissue and ligand are required and the proportion of nonspecific binding may be lower. Lepor and Kuhar were the first investigators to apply this technique for characterization and localization of receptors in the prostate. 9 Using radioligand receptor binding and autoradiographic techniques on slide-mounted tissue sections, these investigators localized prostatic muscarinic cholinergic receptors to the glandular epithelium. The objective of the present study was to characterize and localize human prostatic 3 H-prazosin binding sites using autoradiography and radioligand binding studies on slide-mounted tissue sections. Several investigators have reported that alpha1 adrenoceptors are localized primarily to the Accepted for publication July 7, 1993. * Requests for reprints: Department of Urology, Medical College of Wisconsin, 9200 West Wisconsin Ave., Milwaukee, Wisconsin 53226. This study was supported by NIH Grant 1R29-DK-43149-01.
stromal elements of the prostate. 10- 12 In these studies, the binding assay conditions were not validated, the pharmacology of the binding sites was not characterized and tissue localization was based upon visual inspection of the autoradiograms. In the present study, the optimal preincubation, incubation and washing times were determined, and the Kd, Bmax and pharmacology of these binding sites were characterized using saturation experiments and competitive displacement studies. A quantitative image analysis system was used to localize prostatic alpha1 adrenoceptor binding sites. The development of an efficient, reliable and cost-effective methodology for assaying the pharmacology of prostatic alpha adrenoceptor binding sites will facilitate efforts to identify subtype selective drugs and further characterize the role of these receptors in the pathogenesis and pathophysiology of prostatic diseases. MATERIALS AND METHODS
Tissue specimens. Prostatic tissue was obtained from male subjects with low volume prostate cancer undergoing radical prostatectomies. The surgical specimens were transferred immediately to the surgical pathology laboratory. Prostatic tissue was excised from the benign-appearing elements of the transition zone of the prostate and stored at -SOC. An attempt was made to cut the frozen tissue sections into a rectangular con figuration such that the weight of a single tissue section was estimated by the following equation: (length) X (width) X (thickness) X (tissue density). The prostate tissues were embedded in tissue tech and 20-/.Lm, thick tissue sections were cut using a cryostat set at -20C. The slide-mounted tissue sections were stored at -SOC until the binding studies were performed. Random tissue sections were stained with hematoxylin and
2002
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RADIOLIGAND RECEPTOR BINDING OF ALPHA1 ADRENOCEP'fORS
eosin and inspected to insure the absence of prostatic carcinoma. None of the specimens inspected contained prostate cancer. Assay conditions, incubation intervals. The requirement for preincubation is ligand dependent. 8 Total and nonspecific 3Hprazosin binding were determined at varying preincubation intervals. The tissue sections were allowed to equilibrate to room temperature. Tris buffer (50 mM. Tris HCl, 10 mM. MgCb, pH 7.4) was used for all steps of the assay. The assay was performed in triplicate. Tissue sections were preincubated in buffer for 0, 5, 10, 20, 40 and 60 minutes. The tissue sections were immersed in the incubating solution by pipetting 100 µ,l. of the appropriate stock solution directly over the tissue section. Stock solutions of constant concentrations of 3 H-prazosin (0.5 and 4.0 nM.) were made with and without 4 µ,M. prazosin. The total binding was determined by immersing the prostatic tissue sections in constant concentrations of 3H-prazosin, and nonspecific binding was determined in parallel experiments using the stock solution with 4 µ,M. prazosin. Tissue sections were incubated for 40 minutes at room temperature. Immediately after incubation, the tissue sections were rinsed and then washed twice in buffer for 1 minute. The tissue sections were then wiped off the slides using Q-tips. The Q-tips were immersed in scintillation cocktail overnight, and the total and nonspecific binding were determined from the mean DPMs. The optimal incubation interval was determined by measuring total and nonspecific 3H-prazosin binding at varying incubation intervals of 1 to 60 minutes. The studies were performed at a constant concentration of 3 H-prazosin (0.5 and 4.0 nM.). Washing intervaL The optimal washing time was determined by measuring total and nonspecific 3H-prazosin binding at varying washing intervals. The washing procedure consisted of a brief rinse, 1-minute wash and varying intervals of a second wash in the buffer at room temperature. Saturation studies. Saturation studies were performed at 8 different concentrations (0.0625 nM. to 8.0 nM.) of 3H-prazosin (constant specific activity: 76.2 Ci/mmol.). Nonspecific binding was determined in parallel assays in the presence of a final concentration of 4 µM. cold (nonradiolabelled) prazosin. Total and nonspecific 3 H-prazosin binding were determined in triplicate at each 3H-prazosin concentration. Preincubation was not performed. Incubation was performed by immersing the slide mounted tissue sections with 100 µl. of solution for 40 minutes at room temperature. Immediately after the incubation, the tissue sections were briefly rinsed and washed twice in the buffer for 1 minute. The tissue sections were removed from the slides and the DPM were measured as described above. Competitive displacement studies. Competitive binding experiments were carried out on slide mounted tissue sections in the presence of a constant concentration of 2.0 to 4.0 nM. of 3Hprazosin and varying concentrations of unlabelled prazosin (1 pM. to 1 µM.), phentolamine (10 pM. to 10 µM.) andyohimbine (10 nM. to 100 µ,M.). Tissue localization of alpha1 adrenoceptors. Slide-mounted prostatic tissue sections were brought to room temperature and air dried for 30 minutes. Stock solutions consisting of 3.0 nM. 3 H-prazosin with or without 8 µM. unlabelled prazosin were used to measure total and nonspecific prazosin binding, respectively. Incubation and washings were performed as described for the saturation studies. Immediately after the washing procedure, the tissue sections were dried with a stream of air at room temperature for 60 minutes and stored in a desiccator at room temperature overnight. The tissue sections were apposed to high resolution x-ray film (Hyperfilm- 3H, Amersham, Arlington Heights, Illinois) in cassettes, together with calibrated standards (Autoradiographic [3 H] Micro-Scales, Amersham) for 2, 4, 12 and 20 weeks. The optimal exposure time was 20 weeks. Following a 20-week exposure interval, the film was developed with Dl9 (Kodak, Rochester, New York) at 23C for 3.5 minutes and immersed in an indicator stop bath (Kodak)
for 30 seconds. The film was fixed for 5 minutes and washed in tap water for 30 minutes. Autoradiograms were analyzed quantitatively using the computerized image analysis system (MCID Image Analyzer, Imaging Research, St. Catherine, Ontario Canada). Tissue sections were stained with hematoxylin and eosin to differentiate the stromal and glandular epithelial elements of the prostate. The precise anatomic localization of the alpha1 adrenoceptors was determined by superimposing the microscopic image of the autoradiogram and the histologic image using the MCID Image Analyzer. The region of interest (ROI) was selected from the histological image, and densitometric measurements were subsequently determined from the corresponding ROI of the autoradiogram. The area of the RO Is was approximately 1000 µm. 2 • Optical density was converted to radioactive density (nCi/ mg.) using the standard curve generated from radioactive standards. Regions of interest corresponding to prostatic stroma and glandular epithelium were analyzed separately. The majority of the glandular epithelial elements did not occupy a complete ROI. To obtain reliable radioactive density for epithelium, glandular elements with relatively thick epithelial layers occupying a complete ROI were selected for analysis. Six ROis corresponding to stroma were analyzed from each of the 6 surgical specimens. A total of 4 glands with relatively thick epithelial layers was identified. Six ROis in each of the four glandular epithelial regions were analyzed. Materials. 3H-prazosin (76.2 Ci/mmol.) was obtained from New England Nuclear, Boston, Massachusetts. Prazosin and yohimbine were obtained from Sigma, St. Louis, Missouri. Phentolamine was a gift from Ciba-Geigy. Statistical analysis. The saturation and competitive displacement studies were analyzed using the EBDA computer program.13 The difference between radioactive densities in the stroma and glandular epithelium was examined with the Student t test. A two-tailed probability of less than 5% was taken to indicate statistical significance. RESULTS
Assay conditions. The optimal preincubation interval was determined from two separate experiments. Specific binding was determined for 0.5 and 4.0 nM. 3H-prazosin following preincubation intervals ranging from O to 60 minutes. The plot of a representative experiment demonstrating specific binding versus preincubation interval is presented in figure l. These experiments demonstrated that preincubation is not required. The optimal incubation interval was also determined from two separate experiments. Specific binding was determined for 100
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FIG. 1. Specific 3H-prazosin binding versus preincubation interval in slide-mounted prostatic tissue sections is represented by open circles (0-0-0). Time course for association of specific 3H-prazosin binding in slide-mounted prostatic tissue sections is represented by closed circles (•-•-e).
2004
RADIOLIGAND RECEPTOR BINDING OF ALPHA1 ADRENOCEPTORS 1000
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FIG. 4. Representative competitive displacement curves for specific H-prazosin binding and unlabelled inhibitors prazosin, phentolamine and yohimbine in slide-mounted prostatic tissue sections.
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1. Competitive displacement of /3 H}prazosin binding by
adrenergic inhibitors Adrenergic Inhibitors Tissues
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0.78 ± 0.02 nM.
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l:3H] Prazosin Bound (pM) FIG. 3. A, representative saturation curve demonstrating specific 3H-prazosin binding in slide-mounted prostatic tissue sections. B, representative Scatchard plot for 3H-prazosin binding in slide-mounted prostatic tissue sections.
0.5 and 4 nM. 3H-prazosin following incubation intervals ranging between 1 and 60 minutes. A representative association curve demonstrating specific binding versus incubation interval is shown in figure 1. Specific binding reached a plateau at 40 minutes. The optimal washing time was determined from two separate experiments. A representative washout curve demonstrating
total, nonspecific and specific binding versus washing time is shown in figure 2. Total binding and nonspecific binding were reduced remarkably by a brief rinse and two 1-minute washes. Total and nonspecific binding were not significantly reduced by longer washing intervals. A 1-minutes wash (once or twice) was considered optimal since the ratio of specific binding:nonspecific binding was maximal under these washing conditions. Saturation studies. Saturation studies were performed on tissue sections derived from the central region of 6 radical prostatectomy surgical specimens. Specific 3 H-prazosin binding was determined at 8 different concentrations of 3 H-prazosin ranging from 0.0625 nM. to 8.0 nM. The binding of 3H-prazosin was consistently saturable and of high affinity. The Kd and Bmax were determined from Scatchard analyses of the saturation experiments. A representative saturation curve and Scatchard plot are shown in figure 3, A and B. The mean Kd and Bmax ± standard error determined from 6 saturation studies was 4.16 ± 0.58 X 10- 10 M. and 1.30 ± 0.17 fmol./mg. wet weight, respectively. Competitive displacement experiments. The pharmacology of the 3 H-prazosin binding sites was characterized using competitive displacement experiments. All of the competitive displacement studies were performed at 3 H-prazosin concentrations of 2.0 to 4.0 nM. and varying concentrations of unlabelled prazosin, phentolamine and yohimbine. A total of 3 competitive displacement experiments was performed for each antagonist. A representative competitive displacement plot is shown for the 3 inhibitors (fig. 4). The IC5o corrected (Ki) was determined by analyzing the displacement data using the EBDA computer program. 13 The mean IC 50s corrected for the inhibitors are shown in table 1. Tissue localization of alpha 1 adrenoceptors. Prazosin binding studies were performed on slide-mounted tissue sections to determine the cellular localization of alpha1 adrenoceptors in the human prostate (fig. 5, A). Total and nonspecific 3 Hprazosin binding were determined in the presence of 3 nM. 3 Hprazosin alone and 3 nM. 3 H-prazosin + 8 tiM, unlabelled prazosin, respectively. The autoradiograms representing total
RADIOLIGAND RECEPTOR BINDING OF ALPHA1 ADRENOCEPTORS
2005
3 H-prazosin binding demonstrated localization of the grains predominantly on the prostatic stromal elements (fig. 5, B). Nonspecific binding was uniformly negligible (fig. 5, C). Computer image analysis was used for quantitative analysis of the autoradiograms (table 2). Nonspecific binding accounted for 5 and 21 % of the 3H-prazosin binding in the stroma and glandular epithelium, respectively. Specific radioactive densities were determined by subtracting nonspecific radioactive densities from total radioactive densities. 8 The specific radioactive densities of 3H-prazosin in the stroma and glandular epithelium were 1099 ± 48 pCijmg. and 162 ± 42 pCi/mg, respectively. Specific 3H-prazosin binding was 6.8-fold greater in the stroma than in the glandular epithelium.
DISCUSSION
FIG. 5. A, human prostatic tissue sections stained with hematoxylin and eosin (magnification 20X). B, autoradiogram of human prostatic tissue sections incubated with 3 nM. 3H-prazosin (total prazosin binding) (magnification 20X). C, autoradiogram of prostatic tissue section incubated with 3 nM. 3H-prazosin + 8 µM. unlabeled prazosin (nonspecific prazosin binding) (magnification 20X).
TABLE
2. Tissue localization of prostatic Alpha1 adrenoceptors Radioactive Density (nCi/mg.)
3
H-Prazosin Binding Stroma Total Nonspecific Specific
1162 63 1099
54 8 48
Glandular Epithelium
204 42 162
46 5
42
The present study represents the first characterization of human prostatic alpha1 adrenoceptor binding sites using radioligand receptor binding studies and slide-mounted tissue sections. The optimal assay conditions were determined. The present study demonstrated that preincubation is not required for 3H-prazosin binding studies using prostatic tissue sections. In addition, the optimal incubation interval is 40 minutes, and the optimal washing time is 1 minute (once or twice). The binding of 3H-prazosin to prostatic tissue sections was consistently saturable and of high affinity. Lepor and Shapiro were the first investigators to characterize alpha1 adrenoceptor binding sites in the human prostate using tissue homogenates. 1 These investigators reported that the mean Kd and Bmax of prostatic 3H-prazosin binding sites were 0.29 nM. and 1.12 fmol./mg. wet weight, respectively. The Kd and Bmax values of 3H-prazosin binding sites determined from human prostate homogenates and slide-mounted tissue sections are similar. Competition displacement experiments were performed using the unlabelled antagonists prazosin, phentolamine and yohimbine. Prazosin is a selective alpha1 antagonist, whereas yohimbine is a selective alpha2 antagonist. 1· 14 Phentolamine is relatively nonspecific for alpha1 and alpha2 binding sites. The rank order of the IC 50 corrected values for these inhibitors confirms that alpha1 adrenoceptor binding sites were measured under the assay conditions. We have previously reported that the corrected IC 50s of prazosin, phentolamine and yohimbine in prostatic tissue homogenates were 1.18 x 10-9 , 4.63 X 10-s and 4.08 X 10-6 •4 The competitive displacement studies demonstrate that the pharmacology of the alpha1 adrenoceptor binding sites is similar in tissue homogenates and prostatic tissue sections. Several investigators have reported performing saturation and competitive displacement experiments by immersing the slide-mounted tissue sections in various receptacles. Immersing only the tissue section in the appropriate incubation solutions minimizes the amount of ligand that is required. The cost of 3 H-prazosin required for a single saturation assay using prostatic tissue homogenates and slide-mounted tissue sections is $20 and $4, respectively. These cost savings are even more meaningful when ligands such as 125I-Heat are used to assay alpha1 adrenoceptor binding sites. Several investigators have used radioligand receptor methods and slide-mounted tissue sections to localize prostatic alpha1 adrenoceptors. 10- 12 In these studies, the binding assay conditions were not validated and the pharmacology of the alpha1 adrenoceptor binding sites was not characterized. The binding studies were only performed at a single concentration of the ligands. The present study indicates that the radioligand receptor binding on slide-mounted tissue sections is an appropriate methodology for assaying alpha1 adrenoceptors in the human prostate. Chapple et al. reported that alpha1 adrenoceptors are localized to the prostatic stroma. This conclusion was based solely upon visual inspection of the autoradiograms. The present quantitative tissue localization study confirms the observations reported by Chapple et al. 10 We have previously reported that the ratio of stroma:
2006
RADIOLIGAND RECEPTOR BINDING OF ALPHA1 ADRENOCEPTORS
glandular epithelium in the human prostate is 6.6:1. 16 The present study demonstrated that the relative densities of alpha1 adrenoceptors in the stroma and glandular epithelium are 6.7:1. The proportions of alpha1 adrenoceptor binding sites in the stroma and glandular epithelium of the prostate calculated from these data are 98% and 2%, respectively. The present study establishes the appropriate experimental conditions for characterizing alpha1 adrenoceptor binding sites on slide-mounted human prostatic tissue sections. The present study characterized alpha 1 adrenoceptors only in the transition zone of the prostate. It is conceivable that the binding properties of alpha1 adrenoceptors are different in the various zones of the prostate. A single population of high affinity alpha1 adrenoceptor binding sites was characterized in the transition zone of the human prostate using these methods. The Kd, Bmax and pharmacology of the 3 H-prazosin binding sites were similar to values reported using prostatic tissue homogenates. The present study demonstrates that assaying alpha1 adrenoceptor binding sites directly on prostatic tissue sections is valid and cost- effective, and that 98% of the total alpha1 adrenoceptors are associated with the stromal elements of the prostate. This technique can be applied towards efforts to identify prostaticselective pharmacologic agents and elucidate the pathophysiology and pathogenesis of prostatic diseases. 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. Lepor, H., Baumann, M. and Shapiro, E.: Identification and characterization of alpha 1 adrenoceptor receptors in the canine prostate using 1251-Heat. J. Urol., 138: 1336, 1987. 3. Gup, D. I., Shapiro, E., Baumann, M. and Lepor, H.: Autonomic receptors in human prostate adenomas. J. Urol., 143: 179, 1990. 4. Kobayashi, S.: Characterization and localization of alpha 1 adrenoceptors in human prostate. Jpn. J. Urol., 82: 1241, 1991.
5. Hedlund, H., Andersson, K.-E. and Larson, B.: Alpha adrenoceptors and muscarinic receptors in the isolated human prostate. J. Urol., 134: 1291, 1985. 6. Yamada, S., Ashizawa, N., Ushijima, H., Nakayama, K., Hayashi, E. and Honda, K.: Alpha, adrenoceptors in human prostate: characterization and alteration in benign prostatic hypertrophy. J. Pharmacol. Exp. Ther., 242: 326, 1987. 7. Young, W. S. and Kuhar, M. J.: A new method for receptor autoradiography: 3H-opioid receptors in rat brain. Brain. Rec., 1 79: 255, 1979. 8. Kuhar, M. J. and Unnerstall, J. R.: Receptor autoradiography. In: Methods in Neurotransmitter Receptor Analysis. Edited by H. I. Yamamura, S. J. Enna and M. J. Kuhar. New York: Raven Press, pp. 177-217, 1990. 9. Lepor, H. and Kuhar, M. J.: Characterization and localization of the muscarinic cholinergic receptor in human prostatic tissue. J. Urol., 132: 397, 1984. 10. Chapple, C. R., Aubry, M. L., James, S., Greengrass, P. M., Burnstock, G., Turner-Warwick, R. T., Milroy, J. G. and Davey, M. J.: Characterization of human prostatic adrenoceptors using pharmacology receptor binding and localization. Br. J. Urol., 63: 487, 1989. 11. Kobayashi, S., Demura, T., Nonomura, K. and Koyanagi, T.: Autoradiographic localization of alpha 1 adrenoceptors in human prostate: special reference to zonal difference. J. Urol., 146: 887, 1991. 12. Kawabe, K., Moriyama, N., Hamada, K. and Ishima, T.: Density and localization of alpha 1 adrenoceptors in hypertrophied prostate. J. Urol., 143: 592, 1990. 13. Munson, P. J. and Rodbard, D.: Ligand: a versatile computerized approach for characterization of ligand binding systems. Anal. Biochem., 107: 220, 1980. 14. Shapiro, E. and Lepor, H.: Alpha 2 adrenergic receptors in hyperplastic human prostate: identification and characterization using 3H-rauwolscine. J. Urol., 135: 1038, 1986. 15. Hornung, R., Presek, P., and Glossman, H.: Alpha adrenoceptors in rat brain: direct identification with prazosin. Naunyn-Schmiedebergs Arch. Pharmacol., 308: 223, 1979. 16. Shapiro, E., Hartanto, V. and Lepor, H.: Anti-desmin vs. anti-actin for quantifying the area density of prostate smooth muscle. Prostate, 20: 259, 1992.
Vol. 150, 2002-2006, December 1993
Printed in U.S.A.
CHARACTERIZATION AND LOCALIZATION OF PROSTATIC ALPHA1 ADRENOCEPTORS USING RADIOLIGAND RECEPTOR BINDING ON SLIDE-MOUNTED TISSUE SECTION SHINYA KOBAYASHI, RUI TANG, ELLEN SHAPIRO
AND
HERBERT LEPOR
From the Department of Urology, Medical College of Wisconsin, Milwaukee, Wisconsin
ABSTRACT
Alpha1 adrenoceptor binding sites have been characterized in prostatic tissue homogenates using radioligand receptor binding studies. The objective of the present study was to characterize and localize prostatic alpha1 adrenoceptor binding sites using slide-mounted tissue sections and the ligand 3 H-prazosin. The present study demonstrated that preincubation is not required; the optimal incubation interval is 40 minutes; and a 1-minute wash (once or twice) maximizes the proportion of specific 3 H-prazosin binding. Saturation studies were performed at 8 different concentrations of 3 H-prazosin ranging between 0.0625 nM. to 8.0 nM. The binding of 3 H-prazosin was consistently saturable and of high affinity. The mean Kd and Bmax determined from 6 saturation studies was 4.16 X 10-10 M. and 1.30 fmol./mg. wet weight, respectively. The pharmacology of these 3 H-prazosin binding sites was characterized using competitive displacement experiments. The mean IC 50 corrected for prazosin, phentolamine and yohimbine was 7.8 X 10- 10 M., 6.0 X 10-9 M. and 2.1 X 10-6 M. The rank order of the IC 50 corrected values indicates that alpha1 binding sites were measured under the assay conditions. In the present study, the mean values for Kd, Bmax and IC 50 corrected are similar to values previously reported using prostatic tissue homogenates. Prostatic tissue sections were apposed to x-ray film after being incubated with 3 nM. 3 H-prazosin (total prazosin binding) and 3 nM. 3 H-prazosin + 8 µM. prazosin (nonspecific prazosin binding). The autoradiograms were analyzed using a computerized analyzing system. The specific radioactive densities of 3 H-prazosin in the stroma and glandular epithelium were 1099 ± 48 pCi/mg. and 163 ± 42 pCi/mg. The present study validates the technique of assaying alpha1 adrenoceptor binding sites on slide-mounted prostatic tissue sections and provides further evidence that alpha1 adrenoceptor binding sites are localized primarily to the stromal elements of the prostate. KEY WORDS: receptors, androgen; prostate
Alpha1 adrenoceptor binding sites have been characterized in prostatic tissue homogenates using radioligand receptor binding methods. 1-6 The amount ofprostatic tissue required for a single saturation assay ranges between 1 to 5 gm. owing to a relatively high proportion of nonspecific binding and low capacity of prostatic alpha1 adrenoceptors. 1-6 Gup et al. reported that 1251-Heat is the preferred ligand for characterizing prostatic alpha1adrenoceptors owing to a lower proportion of nonspecific binding. 3 The cost of the ligand for a single saturation assay using 1251-Heat is approximately 5-fold higher than that of 3 H-prazosin. Young and Kuhar and Kuhar and Unnerstall have described a technique for performing radioligand receptor binding studies on slide-mounted tissue sections. 7 • 8 This technique offers several potential advantages for characterizing prostatic atpha 1 adrenoceptors since less tissue and ligand are required and the proportion of nonspecific binding may be lower. Lepor and Kuhar were the first investigators to apply this technique for characterization and localization of receptors in the prostate. 9 Using radioligand receptor binding and autoradiographic techniques on slide-mounted tissue sections, these investigators localized prostatic muscarinic cholinergic receptors to the glandular epithelium. The objective of the present study was to characterize and localize human prostatic 3 H-prazosin binding sites using autoradiography and radioligand binding studies on slide-mounted tissue sections. Several investigators have reported that alpha1 adrenoceptors are localized primarily to the Accepted for publication July 7, 1993. * Requests for reprints: Department of Urology, Medical College of Wisconsin, 9200 West Wisconsin Ave., Milwaukee, Wisconsin 53226. This study was supported by NIH Grant 1R29-DK-43149-01.
stromal elements of the prostate. 10- 12 In these studies, the binding assay conditions were not validated, the pharmacology of the binding sites was not characterized and tissue localization was based upon visual inspection of the autoradiograms. In the present study, the optimal preincubation, incubation and washing times were determined, and the Kd, Bmax and pharmacology of these binding sites were characterized using saturation experiments and competitive displacement studies. A quantitative image analysis system was used to localize prostatic alpha1 adrenoceptor binding sites. The development of an efficient, reliable and cost-effective methodology for assaying the pharmacology of prostatic alpha adrenoceptor binding sites will facilitate efforts to identify subtype selective drugs and further characterize the role of these receptors in the pathogenesis and pathophysiology of prostatic diseases. MATERIALS AND METHODS
Tissue specimens. Prostatic tissue was obtained from male subjects with low volume prostate cancer undergoing radical prostatectomies. The surgical specimens were transferred immediately to the surgical pathology laboratory. Prostatic tissue was excised from the benign-appearing elements of the transition zone of the prostate and stored at -SOC. An attempt was made to cut the frozen tissue sections into a rectangular con figuration such that the weight of a single tissue section was estimated by the following equation: (length) X (width) X (thickness) X (tissue density). The prostate tissues were embedded in tissue tech and 20-/.Lm, thick tissue sections were cut using a cryostat set at -20C. The slide-mounted tissue sections were stored at -SOC until the binding studies were performed. Random tissue sections were stained with hematoxylin and
2002
2003
RADIOLIGAND RECEPTOR BINDING OF ALPHA1 ADRENOCEP'fORS
eosin and inspected to insure the absence of prostatic carcinoma. None of the specimens inspected contained prostate cancer. Assay conditions, incubation intervals. The requirement for preincubation is ligand dependent. 8 Total and nonspecific 3Hprazosin binding were determined at varying preincubation intervals. The tissue sections were allowed to equilibrate to room temperature. Tris buffer (50 mM. Tris HCl, 10 mM. MgCb, pH 7.4) was used for all steps of the assay. The assay was performed in triplicate. Tissue sections were preincubated in buffer for 0, 5, 10, 20, 40 and 60 minutes. The tissue sections were immersed in the incubating solution by pipetting 100 µ,l. of the appropriate stock solution directly over the tissue section. Stock solutions of constant concentrations of 3 H-prazosin (0.5 and 4.0 nM.) were made with and without 4 µ,M. prazosin. The total binding was determined by immersing the prostatic tissue sections in constant concentrations of 3H-prazosin, and nonspecific binding was determined in parallel experiments using the stock solution with 4 µ,M. prazosin. Tissue sections were incubated for 40 minutes at room temperature. Immediately after incubation, the tissue sections were rinsed and then washed twice in buffer for 1 minute. The tissue sections were then wiped off the slides using Q-tips. The Q-tips were immersed in scintillation cocktail overnight, and the total and nonspecific binding were determined from the mean DPMs. The optimal incubation interval was determined by measuring total and nonspecific 3H-prazosin binding at varying incubation intervals of 1 to 60 minutes. The studies were performed at a constant concentration of 3 H-prazosin (0.5 and 4.0 nM.). Washing intervaL The optimal washing time was determined by measuring total and nonspecific 3H-prazosin binding at varying washing intervals. The washing procedure consisted of a brief rinse, 1-minute wash and varying intervals of a second wash in the buffer at room temperature. Saturation studies. Saturation studies were performed at 8 different concentrations (0.0625 nM. to 8.0 nM.) of 3H-prazosin (constant specific activity: 76.2 Ci/mmol.). Nonspecific binding was determined in parallel assays in the presence of a final concentration of 4 µM. cold (nonradiolabelled) prazosin. Total and nonspecific 3 H-prazosin binding were determined in triplicate at each 3H-prazosin concentration. Preincubation was not performed. Incubation was performed by immersing the slide mounted tissue sections with 100 µl. of solution for 40 minutes at room temperature. Immediately after the incubation, the tissue sections were briefly rinsed and washed twice in the buffer for 1 minute. The tissue sections were removed from the slides and the DPM were measured as described above. Competitive displacement studies. Competitive binding experiments were carried out on slide mounted tissue sections in the presence of a constant concentration of 2.0 to 4.0 nM. of 3Hprazosin and varying concentrations of unlabelled prazosin (1 pM. to 1 µM.), phentolamine (10 pM. to 10 µM.) andyohimbine (10 nM. to 100 µ,M.). Tissue localization of alpha1 adrenoceptors. Slide-mounted prostatic tissue sections were brought to room temperature and air dried for 30 minutes. Stock solutions consisting of 3.0 nM. 3 H-prazosin with or without 8 µM. unlabelled prazosin were used to measure total and nonspecific prazosin binding, respectively. Incubation and washings were performed as described for the saturation studies. Immediately after the washing procedure, the tissue sections were dried with a stream of air at room temperature for 60 minutes and stored in a desiccator at room temperature overnight. The tissue sections were apposed to high resolution x-ray film (Hyperfilm- 3H, Amersham, Arlington Heights, Illinois) in cassettes, together with calibrated standards (Autoradiographic [3 H] Micro-Scales, Amersham) for 2, 4, 12 and 20 weeks. The optimal exposure time was 20 weeks. Following a 20-week exposure interval, the film was developed with Dl9 (Kodak, Rochester, New York) at 23C for 3.5 minutes and immersed in an indicator stop bath (Kodak)
for 30 seconds. The film was fixed for 5 minutes and washed in tap water for 30 minutes. Autoradiograms were analyzed quantitatively using the computerized image analysis system (MCID Image Analyzer, Imaging Research, St. Catherine, Ontario Canada). Tissue sections were stained with hematoxylin and eosin to differentiate the stromal and glandular epithelial elements of the prostate. The precise anatomic localization of the alpha1 adrenoceptors was determined by superimposing the microscopic image of the autoradiogram and the histologic image using the MCID Image Analyzer. The region of interest (ROI) was selected from the histological image, and densitometric measurements were subsequently determined from the corresponding ROI of the autoradiogram. The area of the RO Is was approximately 1000 µm. 2 • Optical density was converted to radioactive density (nCi/ mg.) using the standard curve generated from radioactive standards. Regions of interest corresponding to prostatic stroma and glandular epithelium were analyzed separately. The majority of the glandular epithelial elements did not occupy a complete ROI. To obtain reliable radioactive density for epithelium, glandular elements with relatively thick epithelial layers occupying a complete ROI were selected for analysis. Six ROis corresponding to stroma were analyzed from each of the 6 surgical specimens. A total of 4 glands with relatively thick epithelial layers was identified. Six ROis in each of the four glandular epithelial regions were analyzed. Materials. 3H-prazosin (76.2 Ci/mmol.) was obtained from New England Nuclear, Boston, Massachusetts. Prazosin and yohimbine were obtained from Sigma, St. Louis, Missouri. Phentolamine was a gift from Ciba-Geigy. Statistical analysis. The saturation and competitive displacement studies were analyzed using the EBDA computer program.13 The difference between radioactive densities in the stroma and glandular epithelium was examined with the Student t test. A two-tailed probability of less than 5% was taken to indicate statistical significance. RESULTS
Assay conditions. The optimal preincubation interval was determined from two separate experiments. Specific binding was determined for 0.5 and 4.0 nM. 3H-prazosin following preincubation intervals ranging from O to 60 minutes. The plot of a representative experiment demonstrating specific binding versus preincubation interval is presented in figure l. These experiments demonstrated that preincubation is not required. The optimal incubation interval was also determined from two separate experiments. Specific binding was determined for 100
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2004
RADIOLIGAND RECEPTOR BINDING OF ALPHA1 ADRENOCEPTORS 1000
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FIG. 4. Representative competitive displacement curves for specific H-prazosin binding and unlabelled inhibitors prazosin, phentolamine and yohimbine in slide-mounted prostatic tissue sections.
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adrenergic inhibitors Adrenergic Inhibitors Tissues
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l:3H] Prazosin Bound (pM) FIG. 3. A, representative saturation curve demonstrating specific 3H-prazosin binding in slide-mounted prostatic tissue sections. B, representative Scatchard plot for 3H-prazosin binding in slide-mounted prostatic tissue sections.
0.5 and 4 nM. 3H-prazosin following incubation intervals ranging between 1 and 60 minutes. A representative association curve demonstrating specific binding versus incubation interval is shown in figure 1. Specific binding reached a plateau at 40 minutes. The optimal washing time was determined from two separate experiments. A representative washout curve demonstrating
total, nonspecific and specific binding versus washing time is shown in figure 2. Total binding and nonspecific binding were reduced remarkably by a brief rinse and two 1-minute washes. Total and nonspecific binding were not significantly reduced by longer washing intervals. A 1-minutes wash (once or twice) was considered optimal since the ratio of specific binding:nonspecific binding was maximal under these washing conditions. Saturation studies. Saturation studies were performed on tissue sections derived from the central region of 6 radical prostatectomy surgical specimens. Specific 3 H-prazosin binding was determined at 8 different concentrations of 3 H-prazosin ranging from 0.0625 nM. to 8.0 nM. The binding of 3H-prazosin was consistently saturable and of high affinity. The Kd and Bmax were determined from Scatchard analyses of the saturation experiments. A representative saturation curve and Scatchard plot are shown in figure 3, A and B. The mean Kd and Bmax ± standard error determined from 6 saturation studies was 4.16 ± 0.58 X 10- 10 M. and 1.30 ± 0.17 fmol./mg. wet weight, respectively. Competitive displacement experiments. The pharmacology of the 3 H-prazosin binding sites was characterized using competitive displacement experiments. All of the competitive displacement studies were performed at 3 H-prazosin concentrations of 2.0 to 4.0 nM. and varying concentrations of unlabelled prazosin, phentolamine and yohimbine. A total of 3 competitive displacement experiments was performed for each antagonist. A representative competitive displacement plot is shown for the 3 inhibitors (fig. 4). The IC5o corrected (Ki) was determined by analyzing the displacement data using the EBDA computer program. 13 The mean IC 50s corrected for the inhibitors are shown in table 1. Tissue localization of alpha 1 adrenoceptors. Prazosin binding studies were performed on slide-mounted tissue sections to determine the cellular localization of alpha1 adrenoceptors in the human prostate (fig. 5, A). Total and nonspecific 3 Hprazosin binding were determined in the presence of 3 nM. 3 Hprazosin alone and 3 nM. 3 H-prazosin + 8 tiM, unlabelled prazosin, respectively. The autoradiograms representing total
RADIOLIGAND RECEPTOR BINDING OF ALPHA1 ADRENOCEPTORS
2005
3 H-prazosin binding demonstrated localization of the grains predominantly on the prostatic stromal elements (fig. 5, B). Nonspecific binding was uniformly negligible (fig. 5, C). Computer image analysis was used for quantitative analysis of the autoradiograms (table 2). Nonspecific binding accounted for 5 and 21 % of the 3H-prazosin binding in the stroma and glandular epithelium, respectively. Specific radioactive densities were determined by subtracting nonspecific radioactive densities from total radioactive densities. 8 The specific radioactive densities of 3H-prazosin in the stroma and glandular epithelium were 1099 ± 48 pCijmg. and 162 ± 42 pCi/mg, respectively. Specific 3H-prazosin binding was 6.8-fold greater in the stroma than in the glandular epithelium.
DISCUSSION
FIG. 5. A, human prostatic tissue sections stained with hematoxylin and eosin (magnification 20X). B, autoradiogram of human prostatic tissue sections incubated with 3 nM. 3H-prazosin (total prazosin binding) (magnification 20X). C, autoradiogram of prostatic tissue section incubated with 3 nM. 3H-prazosin + 8 µM. unlabeled prazosin (nonspecific prazosin binding) (magnification 20X).
TABLE
2. Tissue localization of prostatic Alpha1 adrenoceptors Radioactive Density (nCi/mg.)
3
H-Prazosin Binding Stroma Total Nonspecific Specific
1162 63 1099
54 8 48
Glandular Epithelium
204 42 162
46 5
42
The present study represents the first characterization of human prostatic alpha1 adrenoceptor binding sites using radioligand receptor binding studies and slide-mounted tissue sections. The optimal assay conditions were determined. The present study demonstrated that preincubation is not required for 3H-prazosin binding studies using prostatic tissue sections. In addition, the optimal incubation interval is 40 minutes, and the optimal washing time is 1 minute (once or twice). The binding of 3H-prazosin to prostatic tissue sections was consistently saturable and of high affinity. Lepor and Shapiro were the first investigators to characterize alpha1 adrenoceptor binding sites in the human prostate using tissue homogenates. 1 These investigators reported that the mean Kd and Bmax of prostatic 3H-prazosin binding sites were 0.29 nM. and 1.12 fmol./mg. wet weight, respectively. The Kd and Bmax values of 3H-prazosin binding sites determined from human prostate homogenates and slide-mounted tissue sections are similar. Competition displacement experiments were performed using the unlabelled antagonists prazosin, phentolamine and yohimbine. Prazosin is a selective alpha1 antagonist, whereas yohimbine is a selective alpha2 antagonist. 1· 14 Phentolamine is relatively nonspecific for alpha1 and alpha2 binding sites. The rank order of the IC 50 corrected values for these inhibitors confirms that alpha1 adrenoceptor binding sites were measured under the assay conditions. We have previously reported that the corrected IC 50s of prazosin, phentolamine and yohimbine in prostatic tissue homogenates were 1.18 x 10-9 , 4.63 X 10-s and 4.08 X 10-6 •4 The competitive displacement studies demonstrate that the pharmacology of the alpha1 adrenoceptor binding sites is similar in tissue homogenates and prostatic tissue sections. Several investigators have reported performing saturation and competitive displacement experiments by immersing the slide-mounted tissue sections in various receptacles. Immersing only the tissue section in the appropriate incubation solutions minimizes the amount of ligand that is required. The cost of 3 H-prazosin required for a single saturation assay using prostatic tissue homogenates and slide-mounted tissue sections is $20 and $4, respectively. These cost savings are even more meaningful when ligands such as 125I-Heat are used to assay alpha1 adrenoceptor binding sites. Several investigators have used radioligand receptor methods and slide-mounted tissue sections to localize prostatic alpha1 adrenoceptors. 10- 12 In these studies, the binding assay conditions were not validated and the pharmacology of the alpha1 adrenoceptor binding sites was not characterized. The binding studies were only performed at a single concentration of the ligands. The present study indicates that the radioligand receptor binding on slide-mounted tissue sections is an appropriate methodology for assaying alpha1 adrenoceptors in the human prostate. Chapple et al. reported that alpha1 adrenoceptors are localized to the prostatic stroma. This conclusion was based solely upon visual inspection of the autoradiograms. The present quantitative tissue localization study confirms the observations reported by Chapple et al. 10 We have previously reported that the ratio of stroma:
2006
RADIOLIGAND RECEPTOR BINDING OF ALPHA1 ADRENOCEPTORS
glandular epithelium in the human prostate is 6.6:1. 16 The present study demonstrated that the relative densities of alpha1 adrenoceptors in the stroma and glandular epithelium are 6.7:1. The proportions of alpha1 adrenoceptor binding sites in the stroma and glandular epithelium of the prostate calculated from these data are 98% and 2%, respectively. The present study establishes the appropriate experimental conditions for characterizing alpha1 adrenoceptor binding sites on slide-mounted human prostatic tissue sections. The present study characterized alpha 1 adrenoceptors only in the transition zone of the prostate. It is conceivable that the binding properties of alpha1 adrenoceptors are different in the various zones of the prostate. A single population of high affinity alpha1 adrenoceptor binding sites was characterized in the transition zone of the human prostate using these methods. The Kd, Bmax and pharmacology of the 3 H-prazosin binding sites were similar to values reported using prostatic tissue homogenates. The present study demonstrates that assaying alpha1 adrenoceptor binding sites directly on prostatic tissue sections is valid and cost- effective, and that 98% of the total alpha1 adrenoceptors are associated with the stromal elements of the prostate. This technique can be applied towards efforts to identify prostaticselective pharmacologic agents and elucidate the pathophysiology and pathogenesis of prostatic diseases. 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. Lepor, H., Baumann, M. and Shapiro, E.: Identification and characterization of alpha 1 adrenoceptor receptors in the canine prostate using 1251-Heat. J. Urol., 138: 1336, 1987. 3. Gup, D. I., Shapiro, E., Baumann, M. and Lepor, H.: Autonomic receptors in human prostate adenomas. J. Urol., 143: 179, 1990. 4. Kobayashi, S.: Characterization and localization of alpha 1 adrenoceptors in human prostate. Jpn. J. Urol., 82: 1241, 1991.
5. Hedlund, H., Andersson, K.-E. and Larson, B.: Alpha adrenoceptors and muscarinic receptors in the isolated human prostate. J. Urol., 134: 1291, 1985. 6. Yamada, S., Ashizawa, N., Ushijima, H., Nakayama, K., Hayashi, E. and Honda, K.: Alpha, adrenoceptors in human prostate: characterization and alteration in benign prostatic hypertrophy. J. Pharmacol. Exp. Ther., 242: 326, 1987. 7. Young, W. S. and Kuhar, M. J.: A new method for receptor autoradiography: 3H-opioid receptors in rat brain. Brain. Rec., 1 79: 255, 1979. 8. Kuhar, M. J. and Unnerstall, J. R.: Receptor autoradiography. In: Methods in Neurotransmitter Receptor Analysis. Edited by H. I. Yamamura, S. J. Enna and M. J. Kuhar. New York: Raven Press, pp. 177-217, 1990. 9. Lepor, H. and Kuhar, M. J.: Characterization and localization of the muscarinic cholinergic receptor in human prostatic tissue. J. Urol., 132: 397, 1984. 10. Chapple, C. R., Aubry, M. L., James, S., Greengrass, P. M., Burnstock, G., Turner-Warwick, R. T., Milroy, J. G. and Davey, M. J.: Characterization of human prostatic adrenoceptors using pharmacology receptor binding and localization. Br. J. Urol., 63: 487, 1989. 11. Kobayashi, S., Demura, T., Nonomura, K. and Koyanagi, T.: Autoradiographic localization of alpha 1 adrenoceptors in human prostate: special reference to zonal difference. J. Urol., 146: 887, 1991. 12. Kawabe, K., Moriyama, N., Hamada, K. and Ishima, T.: Density and localization of alpha 1 adrenoceptors in hypertrophied prostate. J. Urol., 143: 592, 1990. 13. Munson, P. J. and Rodbard, D.: Ligand: a versatile computerized approach for characterization of ligand binding systems. Anal. Biochem., 107: 220, 1980. 14. Shapiro, E. and Lepor, H.: Alpha 2 adrenergic receptors in hyperplastic human prostate: identification and characterization using 3H-rauwolscine. J. Urol., 135: 1038, 1986. 15. Hornung, R., Presek, P., and Glossman, H.: Alpha adrenoceptors in rat brain: direct identification with prazosin. Naunyn-Schmiedebergs Arch. Pharmacol., 308: 223, 1979. 16. Shapiro, E., Hartanto, V. and Lepor, H.: Anti-desmin vs. anti-actin for quantifying the area density of prostate smooth muscle. Prostate, 20: 259, 1992.