Human Hyperplastic Prostate Tissue Research Articles

Adreno-Muscarinic Synergy of Contractile Responses From Human Hyperplastic Prostate.

Adreno-muscarinic synergy, a supra-additional contractile response to simultaneous application of α-adrenoreceptor and muscarinic receptor agonists, is a feature of several lower urinary tract regions that have dual sympathetic and parasympathetic innervation. We tested the hypothesis that synergy is also a feature of prostate tissue obtained from men with benign prostatic enlargement. Isolated tissue strips were dissected from prostate 'chips', collected after transurethral prostate resection procedures for in vitro experiments, to measure isometric tension at 36°C. Added separately to the superfusate, phenylephrine and carbachol generated contractions with mean pEC50 (-log10EC50) values of 5.36 and 5.58, respectively, although phenylephrine maximal responses were about six-fold greater. In the presence of carbachol, the mean phenylephrine pEC50 was significantly increased to 5.84 and maximal response increased by 28%; overall, a significant synergistic response was demonstrated. The synergistic response was reduced by muscarinic receptor antagonists, most potently by the M3-selective agent 4-DAMP (1,1-dimethyl-4-diphenylacetoxypiperidinium iodide), and less so by M2 and M1-selective inhibitors gallamine and pirenzepine, but with an overall profile indicating M3/M2 mediation of the synergistic response. The magnitude of the synergistic response was variable between prostate chips that provided isolated preparations suggesting regional heterogenicity, although their zonal origin could not be determined. These experiments show that adreno-muscarinic contractile synergy is a feature of human hyperplastic prostate tissue. This has implications for the use of a combination therapy of α-blockers and anti-muscarinic agent to relieve secondary symptoms associated with benign prostatic hyperplasia, at least in men who can tolerate antimuscarinics without a risk of retention.

Five-alpha Reductase Inhibitor Influences Expression of Androgen Receptor and HOXB13 in Human Hyperplastic Prostate Tissue

Five-alpha reductase inhibitors (5ARIs) are known as chemopreventive agents in prostate cancer with a risk of high-grade disease. This study evaluated the effects of 5ARI on androgen receptor (AR) and proteins involved in prostate cell growth such as HOXB13 expression in human prostate tissue and LNCaP prostate cancer cells. We retrospectively selected 21 patients who underwent TURP between March 2007 and February 2010 for previously confirmed BPH by prostate biopsy. They were grouped into control (group 1, n = 9) and 5ARI treatment (group 2, n = 12) before TURP. AR and HOXB13 expression in prostate tissue was evaluated by immunohistochemical staining. We tested the effect of 5ARI on the expression of AR, prostate specific antigen (PSA) and HOXB13 in LNCaP cells. Cells were assessed by Western blot analysis, MTT in vitro proliferation assay, and ELISA. Group 2 showed stronger reactivity for AR and HOXB13 than those of the group 1. MTT assay showed death of LNCaP cells at 25uM of 5ARI. At the same time, ELISA assay for PSA showed that 5ARI inhibited secretion of PSA in LNCaP cells. Western blot analysis showed that 5ARI did not greatly alter AR expression but it stimulated the expression of HOXB13. These results demonstrated that 5ARI influences AR and HOXB13 expression in both LNCaP cells and human prostate tissue. In order to use 5ARI in chemoprevention of prostate cancer, we still need to clarify the influence of 5ARI in ARs and oncogenic proteins and its regulation pathway.

Expression and cellular localization of keratinocyte growth factor and its receptor in human hyperplastic prostate tissue.

It is well recognized that the actions of androgens alone do not explain the hyperplastic development of the gland that occurs in elderly men. The increasing number of reports confirming the lack of mitogenic activity of androgens coupled with the powerful mitogenic activity of growth factors and the discovery of growth factor receptors led to an increased interest in the putative role of growth factors in prostate physiopathology. We have previously demonstrated the presence and the cellular localization of epidermal growth factor and of the related peptide, transforming growth factor-alpha, together with their common receptor in the epithelial compartment of the human hyperplastic prostate tissue (BPH). In the present study we examined the expression and cellular localization of messenger ribonucleic acid (RNA) encoding keratinocyte growth factor (KGF) and its receptor in human hyperplastic prostate tissue. RT-PCR of total RNA extracted from BPH tissues documented the presence of transcripts for KGF and its receptor. In situ hybridization with specific RNA probes synthesized from the respective complementary DNA demonstrated that KGF mRNA was mainly localized in the stromal cells, whereas its receptor was mainly localized in the prostate epithelium. Moreover, the mitogenic activity of KGF on cultured BPH cells compared to that of other growth factors has been tested. Our findings clearly indicate that KGF has the ability to function as a potent mitogen in BPH cells. Our data support the hypothesis that KGF plays an important role in prostate growth and that in human prostate it seems to act in a paracrine fashion.

Expression and Cellular Localization of Keratinocyte Growth Factor and Its Receptor in Human Hyperplastic Prostate Tissue

Expression and Cellular Localization of Keratinocyte Growth Factor and Its Receptor in Human Hyperplastic Prostate Tissue

Epidermal growth factor, epidermal growth factor receptor, and transforming growth factor-alpha in human hyperplastic prostate tissue: expression and cellular localization.

It is widely accepted that polypeptide growth factors are involved in the growth and development of normal and neoplastic human prostate. It has been previously reported that epidermal growth factor (EGF) and insulin-like growth factor I (IGF-I) receptors are present in the human hyperplastic prostate tissue (BPH). To add information on the mechanism of action of EGF and transforming growth factor-alpha (TGF alpha), a peptide correlated to EGF, and the EGF receptor (EGF-R) in the human prostate, we studied the expression and cellular localization of messenger ribonucleic acid (RNA) encoding EGF, EGF-R, and TGF alpha in BPH tissue. Reverse transcriptase-PCR of total RNA extracted from BPH tissues documented the presence of specific transcripts for EGF, EGF-R, and TGF alpha. In situ hybridization with specific RNA probes synthesized from the respective complementary DNA demonstrated that EGF, EGF-R, and TGF alpha messenger RNAs were mainly localized in the epithelial cells. Immunprecipitation and Western blot analysis showed that BPH tissue contained the corresponding proteins, EGF and TGF alpha. Our findings provide additional support for the idea that EGF and TGF alpha may be considered specialized symbols in the language of cell-cell interactions and for the hypothesis that in the human prostate they seem to act in an autocrine fashion.

Epidermal growth factor, epidermal growth factor receptor, and transforming growth factor-alpha in human hyperplastic prostate tissue: expression and cellular localization

Epidermal growth factor, epidermal growth factor receptor, and transforming growth factor-alpha in human hyperplastic prostate tissue: expression and cellular localization

G-proteins in alpha 1-adrenoceptor mediated prostatic smooth muscle contraction.

The role of signal transducing guanine-nucleotide binding proteins (G-proteins) in alpha 1-receptor mediated smooth muscle contractions was investigated in human hyperplastic prostatic tissue. The selective alpha 1-receptor agonist phenylephrine (PE) evoked dose dependent contractions antagonized by the alpha 1-receptor blockers prazosin (EC50 10 nM) and YM617 (EC50 3 nM). Application of nifedipine (1-10,000 nM), a blocker of voltage-dependent L-type Ca(2+)-channels (VDCC), inhibited the PE evoked contraction up to 65.4%. Pretreating the tissue strips with pertussis toxin (PTX, exotoxin from Bordetella pertussis; 5-25 micrograms/ml), inactivating a subpopulation of G-proteins, inhibited the PE induced contractions up to 73.9%. PTX pretreatment had no effect on contractions elicited by 125 mM K+. Application of nifedipine to PTX pretreated tissue led to an additional inhibition of 13.7%. Our findings demonstrate the involvement of PTX-sensitive G-proteins in the signal transduction pathway of alpha 1-receptor induced contractions of prostatic smooth muscle. The remaining contractility of PTX pretreated tissue suggests additional participation of PTX insensitive mechanisms in alpha 1-receptor mediated prostatic smooth muscle contractions.

Role of intracellular Ca2+ stores in smooth muscle contractions of the guinea pig vas deferens.

Guinea pig vas deferens was used as an animal model for alpha-1 adrenoceptor (alpha 1-receptor) mediated contractions in human hyperplastic prostatic tissue. The selective alpha 1-receptor agonist, phenylephrine (PE), induced fully reversible, dose-dependent contractions antagonized by increasing concentrations of the alpha 1-receptor blockers prazosin (1-100 nM) and YM 617 (0.1-10 nM). Removal of extracellular Ca2+ reduced PE-evoked contractions in a time-dependent manner. Nifedipine (1-1000 nM), a blocker of voltage-dependent L-type Ca2+ channels (VDCC), inhibited the PE-induced response by up to 65%. Removal of extracellular Ca2+ abolished the alpha 1-agonist reactivity in a time-dependent fashion. To elucidate the participation of intracellular Ca2+ stores in alpha 1-receptor-mediated contractions, the tissue was pretreated with ryanodine (10 microM) or thapsigargin (0.1 microM), established inhibitors of Ca2+ release from intracellular pools. Both substances reduced the PE contractions by up to 80%. Nifedipine suppressed the remaining contractions completely. This provides evidence that Ca2+ influx through VDCC and Ca2+ release from intracellular stores contribute to alpha 1-receptor-mediated contractions in the guinea pig vas deferens and may be important in obstructive benign prostatic hyperplasia.

Insulin-Like Growth Factor-I Receptors in Human Hyperplastic Prostate Tissue: Characterization, Tissue Localization, and Their Modulation by Chronic Treatment with a Gonadotropin-Releasing Hormone Analog*

Insulin-like growth factor I (IGF-I) receptors were characterized in membranes obtained from prostate tissue of patients affected by benign prostatic hyperplasia (BPH) before and after treatment with a GnRH agonist analog. Binding of [125I]IGF-I to membranes obtained from untreated patients was specific and time and temperature dependent. Analysis of the binding data yielded two classes of binding sites, one of high affinity (Kd, 10(-11) mol/L) and one of lower affinity (Kd, 10(-9) mol/L). BPH membrane preparations were affinity-cross linked to labeled IGF-I, and then subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Analysis by autoradiography revealed one labeled protein with an apparent Mr = 300K under nonreducing conditions and two labeled protein with Mr = 270K and Mr = 130K under reducing conditions. Excess unlabeled IGF-II reduce both of them, whereas the same excess of IGF-I completely abolished them. In membrane preparations of prostatic tissues from patients affected by BPH and treated for 2 months with a GnRH agonist analog, the binding capacities of both binding sites were significantly higher than those of BPH tissue from untreated patients, whereas binding affinities were unchanged. The IGF-I receptor in BPH prostate tissue of untreated patients was mainly localized in the basal layer of the epithelium, as demonstrated by immunohistochemical staining, whereas in the tissue from treated patients positive staining was found also in the glandular epithelium. These results demonstrate that: 1) specific binding sites for IGF-I are present in prostatic tissue from patients with BPH, 2) androgen deprivation increases their binding capacities and seems to modify their epithelial localization.

Partial purification of human prostatic 5α-reductase (3-oxo-5α-steroid: NADP + 4-ene-oxido-reductase; EC 1.3.1.22) in a stable and active form

Human hyperplastic prostate tissue was homogenised in high ionic strength buffer and the post nuclear homogenate was incubated with 0.8% octyl glucoside and bovine brain lipids. Dialysis of the resulting liposome suspension yielded a preparation in which 5α-reductase was active and stable for at least three weeks and showed an increase in specific activity ( V max±SD=48.9±7.4pmol DHT/mg protein/ml) over that of the starting homogenate ( V max±SD=5.6±1.5pmol DHT/mg protein/min) of 8.7 times.

Epidermal Growth Factor Receptors in Human Hyperplastic Prostate Tissue and Their Modulation by Chronic Treatment With a Gonadotropin-Releasing Hormone Analog*

We characterized the epidermal growth factor (EGF) receptor in the membrane fraction of prostatic tissue from men with benign prostatic hyperplasia (BPH). The maximum specific binding of [125I]EGF to the BPH membrane fraction was achieved after 30-min incubation at 35 C. Analysis of the binding data revealed two classes of binding sites, one of high affinity [Kd, 2.5 +/- 0.5 (+/- SE) x 10(-11) mol/L] and one of lower affinity (2.2 +/- 0.3 x 10(-9) mol/L). [125I]EGF binding was inhibited by excess EGF, but not by insulin, proinsulin, fibroblast growth factor, or insulin-like growth factors I and II. In prostatic tissue of men with BPH treated for 3 months with the GnRH agonist analog Goserelin (Zoladex, depot formulation), the binding capacities of both sites were significantly higher than those of BPH tissue from untreated men (P less than 0.001). These results demonstrate that prostatic tissue from men with BPH contains two classes of specific binding sites for EGF, and their levels are modulated by chronic GnRH agonists treatment.

Influences of dihydrotestosterone, testosterone, estradiol, progesterone, or prolactin on the cell kinetics of human hyperplastic prostatic tissue in organ culture

In an effort to characterize the hormone sensitivity of human benign prostatic hyperplasia (BPH) maintained in organ cultures for 12-72 h, the influence of 5-alpha-dihydrotestosterone (DHT), testosterone (T), 17-beta-estradiol (E2), progesterone (Pg), or prolactin (PRL) was assessed on the cell proliferation rate of 25 BPH specimens by the use of tritiated thymidine incorporation followed by autoradiography. Significant increases in the thymidine-labeling index (TLI: percentage of labeled nuclei) were observed in glandular tissue after a 36-h incubation period in presence of DHT, E2, Pg, or PRL in 52%, 44%, 28%, and 60% of BPH cases, respectively. Nonparametric statistics (Spearman and Kendall rank correlation tests) have shown that 1) the steroid-induced TLI increases are dependent on the basal rate of cell proliferation, while the PRL-induced effect is independent of it, and 2) all the steroid-mediated effects on BPH TLI are correlated together, whereas they seem to be independent of the PRL-induced TLI increase. When T was compared with DHT on nine BPH specimens, three were found to be sensitive to both hormones, and two responded to DHT only. We propose that our study methods are suitable as a means to assess the hormone sensitivity of individual cases of BPH and possibly prostatic tumors.

36. Measurement of total androgen receptors (AR) in the whole homogenate op human hyper plastic prostatic tissue (BPH) with high concentration cf sodium molybdate

36. Measurement of total androgen receptors (AR) in the whole homogenate op human hyper plastic prostatic tissue (BPH) with high concentration cf sodium molybdate

Studies of the nuclear 5α-reductase of human hyperplastic prostatic tissue

The 5α-reduction of testosterone was studied using the nuclear fraction of hyperplastic human prostatic tissue. Standard assay conditions were: non-radioactive testosterone (4 μM) plus 3.0–4.0 × 10 5 d.p.m. [ 3H]-testosterone, 0.4 mM NADPH, 2.0 mM EDTA and the nuclear fraction equivalent to 200 mg of prostatic tissue, in a final volume of 3.0 ml of 0.1 M HEPES buffer, pH 7.4. Incubations were performed at 37°C for 30 min. Under the conditions of this assay, the conversion of testosterone to androstenedione, dihydrotestosterone (DHT) to 3α-androstanediol (3α Adiol) or 3α Adiol to DHT was negligible. Optimum 5α-reduction was achieved under standard assay conditions. In the absence of EDTA: enzyme activity was 15–25% of the standard assay, 2 mM Ca 2+ or 2mM Zn 2+ further diminished enzyme activity to below 10% of the standard assay, while 2 mM Mg 2+ or 2 μM Zn 2+ increased 5α-reduction to 30% of the standard assay. If NADPH were omitted, 5α-reductase activity virtually was eliminated. NADH (0.4 mM) could not be substituted for NADPH to obtain maximum enzyme activity. The pH optimum of the enzyme was between pH 7.2 and 7.8. The K m of the enzyme was 1.5 μM. The V max, under standard assay conditions, using tissue obtained from 36 men, varied from 30–80 pmol/mg protein/30 min. Cyproterone acetate at a concentration of 1.0 μM had no effect on 5α-reductase activity, but competitively inhibited the enzyme at concentrations of 10 and 100 μM.

14. Distribution of dihydrotestosterone and of nuclear androgen receptors between stroma and epithelium of human benign hyperplastic prostatic tissue (BPH)

14. Distribution of dihydrotestosterone and of nuclear androgen receptors between stroma and epithelium of human benign hyperplastic prostatic tissue (BPH)

Androgen binding in cytosols and nuclei of human benign hyperplastic prostatic tissue.

Androgen binding sites have been detected in cytosols and nuclear extracts from human benign hyperplastic prostatic (BPH) tissue with exchange assays using [3H]methyltrienolone and [3H]5alpha-dihydrotestosterone respectively. The concentrations of binding sites and the equilibrium dissociation constants for the [3H]steroid-receptor interactions have been determined and the specificity of the binding has been examined. The observed properties of the binding sites are consistent with those characteristic of androgen receptors. The binding sites are present in nuclear extracts from all BPH tissue samples examined to date. The amount of binding detected in the nuclear fraction is higher than that found in the cytosol.

Thin-layer chromatography and high resolution selected ion monitoring for the analysis of C19 steroids in human hyperplastic prostate tissue

Interfering nonpolar lipid material was removed from the acetone extracts of several samples of benign hyperplastic prostate tissue. Endogenous steroids were separated by preparative thin-layer chromatography into fractions containing testosterone, 5α-dihydrotestosterone and the 5α-androstane-3, 17-diols, and concentrations of these steroids were measured by molecular ion monitoring of suitable derivatives at a resolution of 8500 to 10 000 during combined gas chromatography mass spectrometry. The sensitivity and specificity of this procedure allowed unequivocal detection of the steroids of interest at the femtomole level and the incorporation of epimers as internal standards enabled accurate quantitative measurements. The results indicated that 5α-dihydrotestosterone is usually the most predominant of the steroids measured, and that of the 5α-androstanediols, only the 3α, 17β and 3β, 17β isomers occur in measurable concentration, with the former usually predominant.

THE INTERACTION OF DIHYDROTESTOSTERONE AND OESTRADIOL-17β WITH MACROMOLECULES IN HUMAN HYPERPLASTIC PROSTATE TISSUE

ABSTRACT During incubation of minced human hyperplastic prostate tissue with [3H]5α-dihydrotestosterone (27 nm), 3H became firmly bound to macromolecules in both cytosol and nucleus. The binding to the cytosol fraction was inhibited when oestradiol-17β (0.3 μm), oestrone sulphate, ethinyl-oestradiol-17β or diethylstilboestrol (10 μm) was included in the incubation. Binding to the nuclei was inhibited by incubation with oestradiol-17β (1 μm) or cyproterone acetate (6.5 μm), a potent anti-androgen, but not by incubation with cortisol (30 μm).

Studies on the interaction between androgen and macromolecules in male accessory sex organs of rat and man

The androgen-receptor complexes in the ventral prostate cytosol fraction are found in different molecular forms, depending on the experimental conditions. By gel fitration two different complexes are found. One is excluded and the other is retained on a column of Sephadex G-100. By experiments in vivo a 6–6.5 S androgen-receptor complex is observed on sucrose gradients. This complex dissociates into a 3.5–4 S complex after dialysis against 0.5 M NaCl (in buffer) for 20 h. The androgen-binding proteins extracted from the nuclear pellet with 0.5 M NaCl move as a sharp peak on sucrose gradients, with a sedimentation constant of 3 S. The epididymis of adult castrated rats also contains similar proteins, which bind 5α-dihydrotestosterone (5α-DHT) with high affinity and low capacity. The 5α-DHT binding protein in the cytosol fraction of epididymal homogenates is slightly retained on a column of Sephadex G-100, and moves with a sedimentation rate of about 4–4.5 S (mean 4.3 S) on sucrose gradients. Similar results are obtained both at high and low ion strength. The binding of [ 3H]5α-DHT to this protein is easily depressed by small amounts of non-labelled 5α-DHT. 15 min after the injection of [ 3H]testosterone in vivo. about 90% of the radioactivity bound to proteins moves as 5α-DHT on t.l.c. 1 h alter the injection of 60 μCi[ 3H]5α-DHT in vivo, a considerable part of the radioactivity is found in the nuclear fraction of homogenized epididymal tissue. Human hyperplastic prostatic tissue also contains similar androgen-binding proteins. The binding of androgens to these macromolecules is inhibited by potent anti-androgenic compounds such as SK & F 7690 and cyproterone. Some evidence for direct binding of [ 3H]cyproterone-acetate in the ventral prostate cytosol fraction is presented.