Ellen Shapiro

Quantifying the Smooth Muscle Content of the Prostate Using Double-Immunoenzymatic Staining and Color Assisted Image Analysis

The primary objective of the present study was to develop a method for quantifying the smooth muscle content of the prostate adenoma. A double immunoenzymatic staining technique was coupled with color assisted image analysis to determine the area density of the smooth muscle within the prostate adenoma. Eight males with symptomatic BPH underwent transrectal biopsy of the prostate. Four micron thick tissue sections were used for the double immunoenzymatic staining process. Rabbit anti-desmin and mouse anti-human prostatic acid phosphatase antibodies were used to selectively bind smooth muscle and prostatic epithelium, respectively. The two different tissue antigens were identified with peroxidase-antiperoxidase (PAP) and alkaline phosphatase-antialkaline phosphatase techniques. The alkaline phosphatase activity and peroxidase activity were developed with fast red and DAB chromogens. The BQ MEG IV Vista color system image analysis was used to discriminate color differences from the stained tissue sections. The thresholds were set to identify smooth muscle (dark brown), epithelium (red), fibrous tissue (pale brown), and glandular lumina (colorless). The mean area density of smooth muscle, fibrous tissue, glandular epithelium, and glandular lumina was 22%, 54%, 16%, and 9%, respectively. The present study suggests that a significant component of the prostate adenoma is smooth muscle. The application of this technique will be utilized to provide further insights into the role of smooth muscle in the pathogenesis and therapy of BPH.

Endothelin-1 in the Human Prostate: Tissue Levels, Source of Production and Isometric Tension Studies

Endothelins mediate contractile responses in many types of vascular and nonvascular smooth muscle. The present study represents the first detailed characterization of endothelins in the human prostate. The objectives of this study were to determine the tissue levels and source of endogenous endothelin-1 (ET1) in the human prostate. The contractile effects of ET1 were also investigated using in vitro isometric tension studies. The mean tissue level of ET1 was 0.58 +/- 0.08 pg./mg. tissue wet weight. Endothelin-like activity was markedly prominent in the glandular epithelium of the human prostate, whereas minimal endothelin-like activity was observed in the prostatic stroma. Strips of human prostatic tissue were suspended in isolated tissue chambers and challenged to a concentration response of ET1. The mean EC50 and Emax for ET1 was 3.2 x 10(-8) M. and 0.12 +/- 0.02 gm. force per mm.2 cross-sectional area (CSA), respectively. Preincubation with indomethacin, terazosin, or nifedipine did not alter the concentration-dependent response to ET1. A calcium-free buffer abolished the contractile response to ET1. Thus, ET1 mediates a potent contraction of human prostatic smooth muscle that is not mediated via alpha 1 adrenergic or dihydropyridine sensitive calcium channels or prostaglandin synthesis. The presence of marked endothelin-like immunoreactivity strongly suggests a biological significance for endogenous endothelins in the human prostate.

Alpha 1 adrenoceptor subtypes in the human prostate.

High affinity alpha 1 adrenoceptors have been characterized in the human prostate. The tension of prostatic smooth muscle is mediated by the alpha 1 adrenoceptor. The present study represents the first characterization of human alpha 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 pM. 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 alpha 1B subtype. The individual displacement plots for WB-4101 and 5-MU in the human prostate were consistently best fit by a 2 binding site model. WB-4101 and 5-MU exhibited a 594- and 186-fold higher affinity for the prostatic alpha 1A binding site relative to the alpha 1B binding site. The ratios of prostatic alpha 1A/alpha 1B binding sites discriminated by WB-4101 and 5-MU were 1.8 and 1.6, respectively. CEC inactivated 44% of the prostatic alpha 1 binding sites. The binding studies suggest that the dominant alpha 1 subtype in the human prostate is the alpha 1A. We are characterizing the functional properties of the alpha 1 subtypes in the human prostate.

Quantitative Morphometry of the Adult Human Bladder

The primary objective of the present retrospective study was to characterize the effects of aging and BPH on bladder morphometry. Eighty-six bladder specimens were obtained from the autopsy archives of the Milwaukee County Medical Complex. The bladder specimens were divided into 4 groups based upon age and gender: Group I: males between the ages of 35-45 years; Group II: males between the ages of 65-75 years; Group III: females between the ages of 35-45 years; and Group IV: females between the ages of 65-75 years. The age groups were selected in order to identify a group of males with and without BPH. The area density of smooth muscle:connective tissue was determined in bladder specimens using color assisted computer image analysis. Masson-trichrome and double immunoenzymatic staining techniques were used to discriminate the smooth muscle and connective tissue elements of the bladder. The area density of smooth muscle:connective tissue in the Masson-trichrome stained sections was significantly greater in Group I vs. Group II (2.90 +/- 0.22 vs. 2.33 +/- 0.16) and in Group III vs. Group IV (2.85 +/- 0.13 vs. 2.03 +/- 0.20). Aging was associated with a decrease in the area density of smooth muscle:connective tissue ratio in both males and females. The area density of smooth muscle:connective tissue was not significantly different in younger males and females (Group I vs. Group III) and older males and females (Group II vs. Group IV). The present morphometric study suggests that aging and not BPH, is associated with a relative increase in detrusor fibrosis. Infravesical obstruction in BPH may effect bladder function via mechanisms unrelated to the histologic composition of the bladder.

Relationship between prostatc epithelial volume and serum prostate-specific antigen levels

Objectives nThe present study was designed to determine the relationship between serum prostate-specific antigen (PSA) levels and prostatic epithelial volumes.

Localization of endothelin receptors in the human prostate

The objective of the present study was to localize endothelin receptors in the human prostate using quantitative autoradiography. Slide-mounted tissue sections 20 microns. in thickness were obtained from the transition zones of seven patients undergoing radical prostatectomies for low volume prostate cancer. Sarafotoxin (S6C) and BQ123 have been used to distinguish endothelin receptor subtypes (ETA and ETB). The prostatic tissue sections were incubated in four different stock solutions containing the following: 0.1 nM. 125I-endothelin-1 (125I-ET-1) (total ET-1 binding); 0.1 nM. 125I-ET-1 and 100 nM. S6C (total ETA binding); 0.1 nM. 125I-ET-1 and 1 microM. BQ123 (total ETB binding); and 0.1 nM. 125I-ET-1 and 1 microM. ET-1 (nonspecific ET-1 binding). Nonspecific binding accounted for only 12 and 15% of total 125I-ET-1 binding in the stroma and glandular epithelium. Autoradiograms were quantitatively analyzed using a computerized image analysis system. Specific radioactive densities (nCi/mg.) were determined for the stromal and glandular epithelial elements of the prostate. The specific radioactive densities of ETA and ETB binding sites in the stroma were 7.57 +/- 0.65 and 2.98 +/- 0.81. The specific radioactive densities of ETA and ETB binding sites in the glandular epithelium were 1.59 +/- 0.15 and 7.87 +/- 1.35. The present study demonstrates that the predominant endothelin receptors in the stroma and glandular epithelium are the ETA and ETB subtypes, respectively.

Characterization and localization of prostatic alpha 1 adrenoceptors using radioligand receptor binding on slide-mounted tissue section.

Alpha 1 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 alpha 1 adrenoceptor binding sites using slide-mounted tissue sections and the ligand 3H-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 3H-prazosin binding. Saturation studies were performed at 8 different concentrations of 3H-prazosin ranging between 0.0625 nM. to 8.0 nM. The binding of 3H-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 3H-prazosin binding sites was characterized using competitive displacement experiments. The mean IC50 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 IC50 corrected values indicates that alpha 1 binding sites were measured under the assay conditions. In the present study, the mean values for Kd, Bmax and IC50 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. 3H-prazosin (total prazosin binding) and 3 nM. 3H-prazosin + 8 microM. prazosin (nonspecific prazosin binding). The autoradiograms were analyzed using a computerized analyzing system. The specific radioactive densities of 3H-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 alpha 1 adrenoceptor binding sites on slide-mounted prostatic tissue sections and provides further evidence that alpha 1 adrenoceptor binding sites are localized primarily to the stromal elements of the prostate.

Investigative Urology: Regional Concentration of Basic Fibroblast Growth Factor in Normal and Benign Hyperplastic Human Prostates

Basic fibroblast grown factor (bFGF) is a potent mitogen for mesenchymal cells, including fibroblasts cultured from prostate, and has been postulated to play a role in the development of benign prostatic hyperplasia (BPH). If this is the case, it might be expected that bFGF levels would be elevated in the adenomas of BPH and in the periurethral region of the prostate where BPH is believed to arise. This study was undertaken to test this hypothesis. The concentration of bFGF was evaluated in 31 prostates, 13 normal glands and 18 with BPH. A method for quantitating bFGF by radioimmunoassay was developed that enabled growth factor levels to be correlated to the geographic region of the prostate and the histopathology of the specimen. A 2- to 3-fold higher concentration of bFGF (ng./g. of tissue) was noted in the benign hyperplastic prostates when compared with the adult normal glands. Pubertal specimens demonstrated low growth factor levels comparable to those observed in the normal adult group. Two prepubertal prostates analyzed had high levels similar to those measured in the hyperplastic glands. While the levels of bFGF in the normal adult prostates were highest in the periurethral region, statistical analysis failed to demonstrate a significant difference. Similarly, quantitative morphometric evaluation failed to demonstrate any significant differences in bFGF concentration related to the proportion of stromal, epithelial, or lumenal elements in the tissue sections.

Binding and functional properties of alpha1 adrenoceptors in different regions of the human prostate.

The objective of the present study was to determine the density and functional properties of alpha 1 adrenoceptors in different regions of the human prostate. Binding and functional studies were performed on eight different topographical regions of the prostate. The contractile response (gm. force/mm.2 cross-sectional area [CSA]) was determined at varying concentrations of phenylephrine, and saturation experiments were performed at seven different concentrations of 125I-Heat. The maximal response to phenylephrine (Emax) ranged from 0.067 to 0.272 gm. force/mm.2 The CSA and the EC50 ranged from 25 to 41 microM. The differences between EC50 and Emax were not significantly different among the eight prostatic regions. A 1.8-fold difference between the Emax for peripheral and central regions of the prostate was statistically significant (p = 0.04). The equilibrium dissociation constant (Kd) of 125I-Heat and the receptor density Bmax were determined from the Scatchard plots. The mean Kd and Bmax ranged from 0.15 to 0.26 nM. and 0.30 to 0.72 fmol. per mg. wet weight, respectively. There were no statistically significant differences between mean Kd and mean Bmax for the eight prostatic regions. The 1.7-fold difference between central and peripheral mean Bmax was not statistically significant (p = 0.07). A direct relationship was not observed between phenylephrine mean Emax and mean Bmax. The present study demonstrates regional differences of the binding and functional properties of prostatic alpha 1 adrenoceptors in the human prostate. These regional differences must be taken into account when investigating the pharmacologic and physiologic properties of the prostate.

Binding and functional properties of alpha 1 adrenoceptors and area density of smooth muscle in the canine prostate.

The present study was designed to compare the area density of smooth muscle, and the binding and functional properties of alpha 1 adrenoceptors in 8 different regions of the canine prostate. The area density of smooth muscle, alpha 1 adrenoceptor density, and contractile response to phenylephrine were investigated using immunoenzymatic staining with color assisted computer image analysis, radioligand receptor binding, and isometric tension studies, respectively. The equilibrium dissociation constants (Kd) for 125I-Heat binding and the alpha 1 adrenoceptor densities (Bmax) in the prostatic regions ranged between 138-230 pM and 0.32-0.52 fmol/wet wt., respectively. The maximal tension generated in the presence of phenylephrine (phenylephrine Emax) and phenylephrine EC50s ranged between 0.043-0.129 gm. force/mm.2 CSA and 4.0-11.0 microM, respectively. The differences between Kd, Bmax, Emax, and EC50 were not significantly different between the different regions of the prostate. The percent area density of smooth muscle ranged between 10.6-24.4%. A direct relationship was not observed between alpha 1 adrenoceptor density and phenylephrine Emax, or alpha 1 adrenoceptor density and percent area density of smooth muscle. A direct relationship was observed between the phenylephrine Emax and percent area density of smooth muscle (p = 0.003; r = 0.90). The phenylephrine Emax and percent area density of smooth muscle was threefold and 1.6-fold greater in the peripheral prostate relative to the central prostate, respectively. The morphometrical and isometric tension studies provides evidence that the canine prostate is a heterogeneous gland.