Pharmacological Study on Human Benign Hyperplastic Prostate: mechanism evaluation of novelα1A? adrenoceptor antagonists and modulation of proliferation of human prostatic smooth muscle cells

• Main Authors: Shih-Chieh J. Chueh, 闕士傑
• Other Authors: Teng Che-Ming
• Format: Others
• Language: en_US
• Published: 2002
• Online Access: http://ndltd.ncl.edu.tw/handle/77244357001195156606

博士 === 國立臺灣大學 === 臨床醫學研究所 === 90 === This thesis explored some new frontiers in the pharmacological studies of benign prostatic hyperplasia (BPH) in humans; which are depicted as follows: We demonstrated that tamsulosin is a potent antagonist against electrical field stimulation-induced and exogenous phenylyephrine-induced contractions in human hyperplastic prostate. The greater potency of tamsulosin against field stimulation induced contractions in BPH tissues is mainly due to the high affinity of tamsulosin for the α1A-adrenoceptor subtype. The results also suggested that tamsulosin had no effect on the voltage-operated calcium channels in human hyperplastic prostate, and tamsulosin had little effect on the noradrenaline release evoked by electrical field stimulation. Some synthesized quinazoline derivatives, namely FH-71, EW-65 and EW-154, were shown to be selective a1-adrenoceptor antagonists. Furthermore, in the functional tension study, we found that FH-71 was the most potent a1A-adrenoceptor antagonist among the mentioned compounds in human hyperplastic prostate. It showed 8- and 19-fold potency than that of EW-65 and EW-154, respectively. These results revealed the developmental potential of FH-71 in the treatment of clinical symptomatic BPH. We showed that the proliferative effects of serum on human prostatic smooth muscle cells (hPSMCs) involved the activation of protein kinase C (PKC), and also characterized the expression of various PKC isozymes in these cells. The association of significant inhibition of serum-induced hPSMCs proliferation by staurosporine and Go-6976, and the significantly increased translocated membrane fraction of PKC-βII during serum-induced cell proliferation suggested that PKC-βII may play a crucial role in serum-evoked proliferative signaling in hPSMCs. We examined the effect of ouabain on the modulation of hPSMCs growth, the predominant factor of BOO in patients with BPH. Ouabain induced hPSMCs proliferation at low concentrations (0.01-1 nM), whereas it produced cytotoxicity of hPSMCs at higher concentrations (10-100 nM). The action of ouabain on hPSMC proliferation was associated with Ca2+ mobilization, and involved the MEK-p42/44 MAPK signaling pathway. Ouabain induced profound hPSMC apoptosis in high concentrations, and caspase-3 was the key executioner of ouabain-induced apoptosis in hPSMCs. We also showed that high concentrations of ouabain induced necrotic cell death as measured by increased LDH release. Keywords: Tamsulosin, Electrical field stimulation, α1-Adrenoceptor subtype, α1-Adrenoceptor antagonist, Human hyperplastic prostate, Smooth muscle cells, Protein kinase C isozymes; Fetal-calf serum, Proliferation, Apoptosis, Ouabain, Caspase. 1. Introduction The prostate gland plays an important role in male reproduction and is also the site of benign and malignant neoplasms. Benign prostatic hyperplasia/hypertrophy (BPH) or benign prostatic enlargement (BPE) refers to the progressive enlargement of the prostate and its increased dynamic adrenoceptor-mediated tone leading to bladder urinary outflow obstruction (BOO) seen commonly and uniquely in aged men. Its symptoms interfere with the normal daily activities, reduce the sense of well-being, and can also be progressive, with a risk of urinary retention, urinary tract infection, bladder calculi, and even renal failure. Endogenous adrenergic stimulation plays an important “dynamic” role in the pathophysiology of BOO in human prostates since the tone of prostatic smooth muscles is significantly increased in the presence of a-adrenergic agonists, and decreased by the a-adrenoceptor antagonists. A dense network of adrenergic nerve fibers has been found within the smooth muscles of the prostatic stromal tissues. That the predominance of a1-adrenoceptors in human prostate primarily mediates its contraction is well recognized. Therefore, one of the main medical treatments for BPH is targeted toward reducing BOO by blocking α-adrenoceptors to relax the tone of prostatic smooth muscles. Further functional and binding studies have shown at least two subtypes of a1-adrenoceptors in human prostate, i.e., α1A- and a1B-adrenoceptor subtypes. With the model of electrical field stimulation, the contractile responses of human prostates were compared with those of rat vas deferens and rat spleen. It is suggested that the α1A-adrenoceptor subtype in human prostates is functionally confined to the synaptic region and it is the major and predominant subtype that mediate contractions to neuronally released noradrenaline. Selective α1A-adrenoceptor antagonists have been developed to optimize the therapeutic effectiveness of relieving BOO in BPH patients and to reduce the adverse effects (e.g. postural hypotension, dizziness, aesthenia) associated with systemic a-adrenoceptor blockade. Tamsulosin is a selective and potent α1-adrenoceptor antagonist which in vitro tension studies and binding assays have shown to function in prostatic tissues. We sought to evaluate the effect of tamsulosin on muscle contraction of human hyperplastic prostates in response to electrical field stimulation in order to define its potency against endogenous adrenergic stimulation of BPH tissues. Accordingly, we have focused our research target on the development of more novel selective α1A -adrenoceptor antagonists that act specifically in prostates. Recently, we have synthesized several quinazoline-based compounds. After the pharmacological characterization, we found that most of these compounds showed a1-adrenoceptor antagonistic properties. In the present work, we examined their a1-adrenoceptor blocking potencies and the selectivity on a1-adrenoceptor subtypes. Furthermore, the therapeutic potentials in the treatment of BPH were also determined in human hyperplastic prostates. BPH is characterized by an androgen-dependent proliferation of prostatic ducts and surrounding stromal tissue. Furthermore, anatomical and pathological studies have shown that stromal heperplasia/enlargement is the first event in the process of BPH and the stromal component composes of 50 ~ 67% of the hyperplastic prostate glands, hense it plays a major role in the pathogenesis of BPH. Shapiro et al. showed that human prostatic smooth muscle cells constitute a major cellular component of prostatic stroma, and their proliferation and increased tension play important roles in BOO secondary to BPH. However, the control of cellular proliferation in human prostates also involves a complex interaction of epithelial and stromal cells. A lot of researches have been conducted to determine the fundamental causes of pathogenesis of BPH. Many factors have been suggested to involve in the development of this disease, including the growth factors and their receptors in the prostate, the regulation of neurotransmitter release, and the hormonal activities of dihydrotestosterone and estrogen. Although serum, insulin and basic fibroblast growth factor have been shown to be mediators of stromal growth in the human prostate, the proliferative mechanisms remain unclear and await detailed investigation. More recently, scientific interest has focused on the reduced cell death theory in prostate and it has been suggested that an imbalance between programmed cell death (apoptosis) and cell proliferation may result in the development of BPH. Protein kinase C (PKC), which is described as a serine/threonine kinase, represents a large gene family of isozymes including Ca2+ -dependent (PKC-α, -βI, -βII and γ) and Ca2+ -independent subfamilies (PKC-δ, -ε, -ζ, -η, -θ and ?λ). These isozymes show differences in their structures, tissue distribution, mode of activation, cofactor dependence, and responsiveness to phospholipid metabolites and substrate selectivity. The existence of so many isozymes exhibits the diversity of PKC-mediated cell responses. However, the functional relevance and their regulation by PKC isozymes appear rather complex and remain incompletely understood. We conducted studies to examine the role of PKC in the regulation of cell proliferation in human prostatic smooth muscle cells (hPSMCs). We also determined which PKC isozymes are present in these cells and characterized their translocations from a cytosolic to a membrane distribution following the stimulation by serum. In our previous reports, we have suggested that ouabain, a cardiac glycoside, could induce an increase in tension response in human prostates, mainly due to an increment in noradrenaline release via an effect on the Na+-dependent Ca2+ influx system. It also increases the resting tone of human hyperplastic prostates following repeated noradrenaline and electrical field stimulation. Ouabain exerts the above dynamic (tension-related) roles in the concentration range of more than micromolar levels. Of note, the therapeutic blood concentration of cardiac glycosides is nanomolar levels or less, and this range can be easily reached in cardiac glycoside-administered patients. Furthermore, circulating endogenous inhibitors of the plasmalemma Na+ pump, i.e., the ouabain-like substance, could be responsible for increased vascular smooth muscle tone in some forms of hypertension, or it is also released in response to an excessive salt intake. It shows that the Na+-K+ ATPase inhibitor, either the therapeutic cardiac glycoside or the endogenous ouabain-like substance, may be a causative factor in some diseases, such as hypertension. However, it is interesting for us to know if the therapeutic concentrations of ouabain plays a role, especially a static (proliferative, space-occupying) role, in the pathophysiology of BPH. To examine this idea, we cultured hPSMCs from different patients with BPH as the experimental model and determined the effect of ouabain on the regulation of cell growth, and also investigated its detailed mechanisms. 2. Materials and methods 2. 1. Human prostatic tissues Human hyperplastic prostates were obtained at operation from symptomatic BOO-associated BPH patients. All these patients were diagnosed to have BPH by histopathology of the prostatic specimens and evaluated preoperatively with the combinations of lower urinary tract symptoms, digital rectal examination (DRE, no hard nodule), serum prostatic specific antigen (PSA, < 4 ng/ml), transrectal ultrasonography of the prostate, and urodynamic studies. The protocol of this study complies with the Declarations of Helsinki and Tokyo for humans. The specimens were used for in vitro isometric tension experiments and primary culture of human prostatic smooth muscle cells. 2.2. Rat aortae, vas deferens, and spleens The protocol of this study complies with the European Community guidelines for animals. Male Wistar rats (250-300 g) were sacrificed, then the thoracic aortae, vas deferens and/or spleens were harvested. The vessels (denuded and then cut into rings of about 5 mm in length), the vas or spleen (hemisected) was then mounted and equilibrated under the same conditions as human hyperplastic prostates for 90 min under a resting tension of 1 g (0.5 g for vas). Similar protocols as in human prostates were carried out to evaluate the potencies of indicated inhibitors in rat aortae. After the equilibration period, rat vas deferens were contracted twice with 10 mM phenylephrine and then washed and equilibrated for a further 30 min. Non-cumulative concentration-response curves for phenylephrine-induced contractions were determined in the absence or presence of the indicated concentrations of antagonists. 2.3 Novel drugs in development A series of quinazoline derivatives was designed and synthesized as potential a1A-adrenoceptor antagonists by Dr. Ji-Wang Chern (unpublished results) and FH-71 (Ethyl 4-(3-(4-(2-methoxyphenyl) piperazinyl)aminoquinazolin-2-carboxylate), EW-65 (4-(3-(4-(2- methoxyphenyl)piperazinyl)propyl)aminoquinazolin-2-carboxamide) and EW-154 (2-(4-(4-(2-methoxyphenyl)piperazinyl)butyl)amino-4- cyclohexylaminoquinazolin) (Fig. 3-1) were studied in the present work. 2.4 In Vitro isometric experiments Immediately after removal, the prostatic specimens were cut into strips and mounted in a 37oC organ bath containing gassed Krebs solution. The tissues were equilibrated for 90 min under an optimal resting tension of 0.5-1 g before specific experimental protocols were initiated. Contractions were recorded isometrically via a force-displacement transducer (Grass, model 7DAG) connected to a Grass polygraph. Cumulative concentration-response curves (CRC) for agonist-induced contractions were determined in the absence or presence of the indicated antagonists. The tissues were equilibrated with each antagonist for 15 min before each CRC was obtained. Four reproducible CRCs were made for a given antagonist. For electrical field stimulation, the tissues were mounted vertically into two parallel platinum ring electrodes in organ baths. Intramural nerve stimulation was performed by means of an electronic stimulator (Grass model S88, 0.3 ms duration at 80 V and 20 Hz for 5 s). The almost complete inhibition of the response by tetrodotoxin (0.1 μM) confirmed that the contractions induced by transmural stimulation were nerve-mediated (n=8). The contractile effect of calcium was evaluated in a high-K+ (60 mM) solution without Ca2+. The tissues were incubated in KCl (60 mM) / Ca2+ -free medium in the presence of prazosin (10-7 M, to block α1- adrenoceptor-mediated responses) and then incubated in the absence or presence of tamsulosin (10-9 M) or nifedipine (10-5 M) at 37oC for 15 min. Cumulative concentrations of Ca2+ (10-4 to 3 × 10-3 M) were then used to evoke contractions. 2.5 Release of [3H]noradrenaline from prostatic strips The tissues were loaded with 1-(7,8)- [3H]noradrenaline (3 μCi /ml) for 60 min at 37oC in gassed Krebs solution, then washed with Ca2+-free Krebs solution containing 0.04 mM EDTA for 90 min. Then they were incubated in Krebs solution for a further 20 min. After the above experimental procedure, the tissues were incubated in Krebs solution, and the solution was changed every 3 min and collected. From the amount of tritium in the tissue and in the efflux sample, fractional rate of loss (FRL, per 3 min) of tritium were calculated, i.e., the amount of tritium which appeared in the solution during any given 3 min collection period was expressed as a fraction of the amount of tritium present in the tissue at the beginning of the respective collection period. At the end of the experiments, the tissues were homogenized, extracted overnight, and centrifuged. The tritium content of the extracted supernatant and of the collection samples was determined. During the experiments, the tissues were stimulated electrically (0.3 ms duration at 80 V and 20 Hz for 5 s) three times: at the beginning of the 3rd, 13th and 23rd collections. The second stimulation (S2) was used as a control; the third stimulation (S3) was performed in the absence or presence of the drug applied. The drug used was added to the solution 15 min before S3. Effects of drugs were expressed as the ratio S3/S2 of the overflow of tritium evoked by the two stimulation periods. 2.6 Tissue explants and subcultures Prostatic tissue explants were managed and cultured cells were obtained as previously (Guh et al., 1998). Isolated human prostatic smooth muscle cells (hPSMCs) were identified as previously by the following criteria: the cultured cells exhibited positive immunofluorescence staining for vimentin and smooth muscle a-actin, whereas showed negative immunostaining with epithelial cytokeratins; the culture morphology was characterized by the formation of nodules of cells, that is, 'hills and valleys'. 2.7 Cell proliferation assay The cell proliferation and/or cytotoxic assay of the mentioned agents were carried out using the MTT assay described by Mosmann (1983). 2.8 Electrophoresis and Western blotting analysis HPMSCs in each treatment group were isolated from culture dishes and suspended in buffer A (4oC), then cells were ultrasonically disrupted, and lysates were centrifuged (100000 × g for 1 h). The soluble cytosolic fraction was retained and the membrane pellet was resuspended in 0.3 ml of buffer A and sonicated. Protein concentration of each cytosolic and membrane fraction was determined by the Bio-Rad protein assay. All fractions were then diluted with loading buffer and boiled for 5 min. For Western blot analysis, the amounts of cytosolic (20 μg) and membrane (40 μg) protein were fractionated in 9% polyacrylamide gel electrophoresis in the presence of 0.1% SDS, according to Laemmli (1970). Proteins were then electroplotted onto nitrocellulose membranes. The membranes were prepared and then incubated overnight at 40C with diluted anti-protein kinase C-α, -βI, -βII, -γ, -δ, -ε, -ξ, -η -θ, and -λ isozyme-specific antibodies. After four washings, alkaline phosphate-conjugated anti-mouse or anti-rabbit immunoglobulin G (IgG) was applied to the membranes for 1 h at room temperature. The membranes were washed and then incubated in alkaline phosphatase buffer at room temperature for 10 to 20 min. Alkaline phosphatase was finally detected in visible blots. Measurement of phosphorylation of p42/44 MAPKs. Activation of p42/44 MAPKs in cultured hPSMCs was determined by Western blot using a rabbit polyclonal antibody raised against dually phosphorylated p42/44 MAPKs. For Western blot analysis, cell lysates (25 mg.lane-1) were electrophoresized on 15% SDS-polyacrylamide gels, and transferred to a nitrocellulose membrane. The membranes were probed with anti-phosphorylated MAPK polyclonal antibody, which binds to p42/44 MAPKs when they are activated by phosphorylation at Thr-202 and Tyr-204. The same samples of each measurement were also probed with a polyclonal antibody detecting non-phosphorylated p42/44 MAPKs to ensure equal loading of the samples. The transferred membranes were developed with a secondary anti-rabbit antibody as previously described. 2.9 Apoptosis and necrosis assays Preparation of cytosolic extracts and measurement of caspase-3 activity. After the treatment of ouabain, hPSMCs were washed twice with ice-cold PBS, and then collected by centrifugation. The cell pellet was resuspended in lysis buffer (25 ml., 106cells-1) obtained from a commercial assay kit (Caspase-3 colorimetric assay kit, R&D Systems Inc.). After 10 minutes incubation on ice, cell homogenates were centrifuged at 10,000xg for 1 minute and supernatants were removed for the determination of caspase-3 activity. Proteolytic reactions were performed in a total volume of 100 ml. reaction buffer containing 50 ml. of cytosolic extracts and 5 ml. DEVD-pNA obtained from the same commercial kit described above. The reaction mixture was incubated at 37oC for one to two hours, and then the formation of p-nitroanilide was measured at 405 nm. by an ELISA reader. In situ labeling of apoptotic cells. In situ detection of apoptotic cells was carried out by using a terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling (TUNEL) method with an apoptotic detection kit. This method identifies apoptotic cells in situ by using TdT to transfer biotin-dUTP to the free 3’-OH of cleaved DNA. The biotin-labeled cleavage sites then were visualized by reaction with fluorescein conjugated avidin (avidin-fluorescein isothiocyanate). Photomicrographs were obtained with a fluorescence microscope (Nikon). Assessment of cell necrosis. The necrotic cell death was measured by the release of lactate dehydrogenase (LDH) into the culture medium, which indicates the loss of membrane integrity and cell necrosis. LDH activity was measured using a commercial assay kit (Cytotoxicity assay kit, Promega, Madison, WI, USA), where the released LDH in culture supernatants is measured with a coupled enzymatic assay which results in the conversion of a tetrazolium salt into a red formazan product. The necrotic percentage was expressed as (sample value/maximal release) x 100%, where the maximal release was obtained following the treatment of control cells with 0.5% Triton X-100 for 10 minutes at room temperature. 2.10 Data analysis In each experiment, agonist-elicited concentration-response curves in the presence of the indicated concentrations of each antagonist were related to the control concentration-response curve of which the maximum response of which was taken as 100%. The concentration of the agonist (phenylephrine) necessary to give a half-maximum response in the presence of each concentration of antagonist was divided by the concentration giving a half-maximum response in the absence of antagonist to determine the dose ratio (DR). The data were plotted by the method of Arunlakshana and Schild (1959) as the ? log (antagonist concentration, M) vs. the log (DR ?1); when the DR 2, the ?log (antagonist concentration) was taken as pA2 value from the Schild plot (Mackay, 1978). Additionally, the concentration of antagonist needed to produce half-maximal inhibition (IC50) in the absence of antagonist was determined, and the ?log (antagonist concentration) was taken as pIC50 value. The experimental results are expressed as means ± S.E.M. and are accompanied by the number of observations. Statistical analysis of data was evaluated by unpaired two-tailed Student's t-test or with one-way analysis of variance (ANOVA) followed by a t-test and P values of less than 0.05 were considered significant. 3. Results 3.1 Inhibition by tamsulosin of tension responses of human hyperplastic prostate to electrical field stimulation l Phenylephrine-induced responses Prazosin and tamsulosin caused concentration-dependent parallel rightward shifts of the concentration-response curve of phenylephrine in human hyperplastic prostate tissue (n=5). Schild plots were constructed for the effects of prazosin and tamsulosin at various concentrations. The slopes of these regressions did not differ significantly from negative unity (for prazosin, -1.14 ± 0.06 ; for tamsulosin, -1.19 ± 0.02). The pA2 values were calculated to be 9.25 ± 0.07 and 10.05 ± 0.16 for prazosin and tamsulosin, respectively; the relative potency of tamsulosin with reference to prazosin was 6.31. Electrical field stimulation-induced responses The contractile responses to electrical field stimulation in human hyperplastic prostate were concentration dependently blocked by pretreatment with tamsulosin (10-10- 10-9 M) or prazosin (10-9- 10-8M). The IC50 values of prazosin and tamsulosin against field stimulation were (2.11 ± 0.21) × 10-9 M and (1.93 ± 0.26) × 10-10 M, respectively and the pIC50 values were 8.69 ± 0.04 and 9.73 ± 0.05 (n=4), respectively. The relative potency of tamsulosin with reference to prazosin was 10.96. Effects of tamsulosin and nifedipine on high K+-induced Ca2+ dependent contraction In the presence of prazosin (10-7 M) to block α1?adrenoceptor responses, the cumulative addition of Ca2+ (10-4 to 3× 10-3 M) caused a stepwise increase of muscle tension in isolated human hyperplastic prostate, which was pre-depolarized with 60 mM K+ in a Ca2+-free medium. The maximum tension attained at 3×10-3 M Ca2+ was 0.54 ± 0.04 g and was taken as 100%. Tamsulosin (10-9 M) had little effect on this Ca2+ -induced muscle contraction under the above conditions. The EC50 value of calcium in the presence of tamsulosin was (8.2 ± 0.4) × 10-4 M compared to a control value of (7.4+0.3) x 10-4 M which is not significantly different. However, nifedipine (10-5 M) almost completely abolished this Ca2+ -induced contraction in human hyperplastic prostate. Effects of prazosin and tamsulosin on electrical field stimulation-evoked [3H] noradrenaline release Electrical field stimulation significantly evoked the release of [3H]noradrenaline, which amounted to 5.3 ± 1.0%, 4.8 ± 0.9% and 4.5 ± 1.1%, respectively for the first (S1), second (S2) and third (S3) stimulation. The S3/S2 ratio was calculated and was used to examine the effect of drugs on [3H] noradrenaline release. The S3/S2 ratios were 0.94 ± 0.01, 0.96± 0.02 and 0.90 ± 0.02 (n=4) for dimethyl sulfoxide (0.1%, control), prazosin (10 nM) and tamsulosin (1 nM), respectively, indicating that both prazosin and tamsulosin had little effect on the [3H] noradrenaline release evoked by electrical field stimulation in human hyperplastic prostates. 3.2 Characterization of some novel a1-adrenoceptor antagonists in human hyperplastic prostate Examination of the selectivity on a1-adrenoceptors The a1-adrenoceptor antagonistic properties of FH-71, EW-65, and EW-154 were evaluated against phenylephrine-induced contractions in rat thoracic aorta. In a low concentration (0.1 mM), all of these compounds reversibly shifted the concentration-response curves to phenylephrine; whereas they showed little influence on those to U46619 (a thromboxane A2 receptor agonist) and high K+ (even in a high concentration of 10 mM) suggesting the selectivity of these antagonistic agents on a1-adrenoceptors. The potencies of these compounds against phenylephrine-induced contraction in rat aorta were determined. The results showed that FH-71, EW-65 and EW-154 all caused concentration-dependent parallel rightward shifts of the concentration-response curves to phenylephrine in a competitive manner. Schild plots were constructed and the pA2 values were calculated to be 8.22, 8.37, and 7.16, respectively. Determination of a1-adrenoceptor subtype potency in rat vas deferens and spleen There are several lines of evidence suggesting that contractions in response to noradrenaline are mediated predominantly by a1A-adrenoceptor subtypes in rat vas deferens, and by a1B-adrenoceptor subtypes in rat spleens. In the present work, the contractions in rat vas deferens and spleens elicited by phenylephrine were used as models for a1A- and a1B-adrenoceptor subtypes, respectively. The results showed that terazosin, FH-71, EW-65 and EW-154 all caused concentration-dependent parallel rightward shifts of the concentration-response curves to phenylephrine in a competitive manner without diminishing the maximal contractions in rat vas deferens and spleens. Schild plots were constructed from the effects of the above a1-adrenoceptor antagonists at various concentrations; the pA2 values were calculated and the relative potencies of FH-71, EW-65 and EW-154 with reference to terazosin were also determined (pA2 =8.12, 8.62, 7.15, 6.68 in vas deference; and 8.40, 7.62, 7.84, 8.02 in spleen, respectively). The data showed that FH-71 was the most potent against the action to phenylephrine in rat vas deferens, whereas the least effective to that in rat spleens. On the contrary, EW-154 was much more potent to that in rat spleens other than vas deferens. And EW-65 had similar effects in both tissues. The potency ratios of these compounds against a1A- and a1B-adrenoceptor subtypes were also calculated. With reference to terazosin (as 1), FH-71 had a 19-fold selectivity in a1A- than a1B-adrenoceptor subtypes; with contrast, EW-154 exhibited a 11-fold selectivity in a1B- than a1A-adrenoceptor subtypes. Examination of a1-adrenoceptor antagonistic potency in human hyperplastic prostate Phenylephrine induced a contractile response in human hyperplastic prostates in a concentration-dependent manner; the maximum contraction was 0.82 ±0.06 g (n = 16). Terazosin, FH-71, EW-65 and EW-154 all caused concentration-dependent parallel rightward shifts of the concentration-response curve of phenylephrine in hyperplastic prostates, and the Schild plots were constructed. The pA2 values were calculated from the Schild plots (8.17, 8.34, 7.44, 7.05, respectively) and it showed that FH-71 was the most potent one against the contraction to phenylephrine action in human prostates. Determination of the cytotoxic effects in human prostatic smooth muscle cells We also determined the cytotoxic effects of these compounds in cultured human prostatic smooth muscle cells. FH-71 and EW-65 had little cytotoxic effects using MTT assay methods; however, EW-154 (10 mM) induced a significant cytotoxicity (more than 80%) in these cells. Furthermore, using lactate dehydrogenase release assay, we also found that EW-154 but not FH-71 and EW-65 induced a profound increase of LDH release reaction suggesting the necrotic other than apoptotic effect to EW-154 action. 3.3 Cell proliferation in human prostatic smooth muscle cells involves the modulation of protein kinase C isozymes MTT assay suitable for measuring hPSMCs numbers The correlation between cell number and absorbance density value for MTT assay of human prostatic smooth muscle cells (hPSMCs) was measured in this study. It showed that there was a proportional increase in the absorbance density values parallel to the increase in cell number with an r value of 0.99. These results indicate a very good correlation between the absorbance density value and cell number and show that this MTT assay is suitable for the measurement of cell proliferation. Effects of PKC inhibitors on hPSMC proliferation To study the proliferative action in hPSMCs, cultured cells were made quiescent by the deprivation of serum for 48 h. The reintroduction of fetal-calf serum (10%, v/v) induced a significant cell proliferation by MTT assay method (91.3 + 8.7% increase over control). To investigate the involvement of protein kinase C (PKC)-signaled pathway, two PKC inhibitors, staurosporine and Go-6976 [12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo(2,3-a)pyrrolo(3,4-c)-carbazole, a selective Ca2+-dependent PKC inhibitor], were used in this proliferative action to serum in this study. Both staurosporine and Go-6976 (10-100 nM) produced significant reductions in serum-stimulated hPSMC proliferation. However, at the concentration of 100 nM, staurosporine but not Go-6976 had cytotoxic effect on these cells (71.0 + 4.9 and 95.5 ± 3.1% survival as compared with the pretreatment control, respectively). Expression of PKC isozymes in hPSMC proliferation We have examined the expression of PKC isozymes in hPSMCs using Western blot technique. The results showed that Ca2+-dependent protein kinase C-α, βI, and βII, were the predominant isozymes present in human prostatic smooth muscle cells. In addition, protein kinase C-δ, -ε and -ξ were also found to be present but in less abundant concentrations; however, protein kinase C-γ, -η, -θ, and -λ were not detected in these cells. Translocation of PKC isozymes in hPSMC proliferation To investigate which PKC isozyme plays a crucial role in the regulation of PKC-signaled proliferation, hPSMCs were treated with 10% fetal-calf serum for a different time course (1 to 45 min) to induce the redistribution of PKC isozymes in these cells. Fetal-calf serum induced a rapid translocation of PKC-βII and PKC-ε from the cytosolic to the membrane fraction in these cells; by contrast, PKC-α, -βI, -δ and -ξ were also examined but remained at unchanged levels in both cytosolic and membrane fractions regardless of serum treatment. 3.4 Dual effects of ouabain on the regulation of proliferation and apoptosis in hPSMCs Effect of ouabain on hPSMC proliferation and the regulation by Ca2+/calmodulin. To investigate the proliferative action of ouabain on hPSMCs, cultured cells were made quiescent by the deprivation of serum for 48 hours. Ouabain of various concentrations was added into the cultures for another 24 hours, and MTT assay was performed. At concentrations of 0.01 to 1 nM, ouabain modestly yet significantly increased the cell number of hPSMC as compared with control (quiescent cells without ouabain). The greatest number of cells was achieved with 0.1 nM of ouabain (116 ±1.7% with respective 110, 112, 121, 130, 109, 121, and 109% increase of control for 7 patients). However, in the higher concentrations (more than 10 nM), ouabain induced the decrease other than the increase of cell number suggesting the occurrence of cell death. It has been shown that the presence of extracellular Ca2+ and/or Ca2+ mobilization was required for several ouabain-induced cellular functions. In this study, the experimental design of extracellular Ca2+-free medium was impracticable in the functional assay. The Ca2+/calmodulin antagonist, W-7, was used to examine if the Ca2+ mobilization and calmodulin were required in this ouabain action. The results demonstrated that W-7 (10 mM) almost completely abolished the proliferative effect of ouabain as measured by MTT assay suggesting that the Ca2+ mobilization and calmodulin shared the signal pathways in ouabain-induced hPSMCs proliferation. Effect of ouabain on p42/44 MAPK activation. Involvement of p42/44 MAPK isoforms (ERK1 and ERK2) in the regulation of cell growth has been suggested in a lot of studies and activations of these MAPKs by growth factors and pharmacological agents have also been demonstrated. In this study, we have examined whether these kinases are activated when hPSMCs are exposed to ouabain that causes the proliferation in these cells. The levels of phosphorylated and activated p42/44 MAPKs were determined using Western blot analysis after the exposure of hPSMCs to ouabain for several time courses. Ouabain (1nM) increased the phosphorylation of both p42 and p44 MAPKs in a time-dependent manner. When concentration-dependent changes of ouabain were examined, significant increase of the phosphorylation of p42/44 MAPKs were detected with low concentrations of ouabain (0.03 to 1 nM); however, the phosphorylated p42/44 MAPKs were dramatically declined when the cytotoxic concentrations (10-100 nM) of ouabain were used. Effect of MEK inhibitor on ouabain-induced effect. To further investigate the possible signal pathways involved in ouabain-induced proliferation of hPSMCs, PD98059, a highly specific inhibitor of MEK, was used to verify the role of MEK in ouabain-induced effect. The addition of PD98059 (10 mM) significantly inhibited ouabain-induced phosphorylation of p42/44 MAPKs in hPSMCs. Effect of ouabain on apoptosis by TUNEL assay. The hallmarks of apoptosis are nuclear chromatin condensation and fragmentation of DNA, which can be visualized in situ by the TUNEL-reaction by labeling breaks in the DNA strand. We examined if higher concentrations of ouabain induced apoptosis in hPSMCs by TUNEL assay. The control cells showed negative staining, whereas ouabain (100nM) significantly induced the cell apoptosis and the positive TUNEL-reaction was observed as the strong green fluorescence staining. The increase of caspase-3 activity by ouabain. To determine whether caspase-3 executed the apoptotic action by ouabain, various concentrations of the peptide inhibitor of caspase-3, DEVD-CHO, were used in this experiment. The data showed that DEVD-CHO diminished the ouabain (100 nM)-induced apoptosis in a concentration-dependent manner (0-300 nM). Moreover, we have also measured the caspase-3 activity after the exposure of cells to ouabain. The results showed that ouabain significantly and concentration-dependently (10 to 100 nM) increased the caspase-3 activity in hPSMCs. Furthermore, the release of LDH, an indicator of cell necrosis, was also assayed for the measurement of cell necrosis by ouabain. It showed that at high concentrations of 10 and 100 nM, ouabain also significantly induced the cell necrosis in hPSMCs. 4. Discussions Tamsulosin has potent antagonistic effect on human prostate in the in vitro functional study with externally applied noradrenaline (Chapple et al., 1994a). However, its effect on noradrenaline released on nerve stimulation, the real pathophysiology of BPH, has not been documented before. We examined the effect of tamsulosin on contractile responses to phenylephrine and electrical field stimulation in human hyperplastic prostate. The relative potencies of tamsulosin with reference to prazosin were calculated and compared, since prazosin exhibited no selectivity for α1-adrenoceptor subtypes (Hanft and Gross, 1989). The results showed that tamsulosin exhibited greater potency against field stimulation-induced contraction compared to that against phenylephrine. Guh et al. (1995) has suggested that the major subtype mediating contractions to neuronally released noradrenaline in human prostate is the α1A ?adrenoceptor subtype; additionally, the contraction in response to exogenously applied noradrenaline is mediated by both α1A ? and α1B ?adrenoceptor subtypes (Teng et al., 1994). Michel and Insel (1994) demonstrated that the affinity of tamsulosin for the α1A?adrenoceptor subtype was ten times that for the α1B ?adrenoceptor subtype. It is suggested that the greater potency of tamsulosin against field stimulation induced contractions in human hyperplastic prostate is due to the high affinity of tamsulosin for the α1A-adrenoceptor subtype. The α1A ?adrenoceptor subtype requires the influx of extracellular Ca2+ through dihydropyridine-sensitive channels to cause smooth muscle contraction (Minneman, 1988). In the present study, tamsulosin (10-9 M) had no effect on high K+ (60 mM) ?depolarized Ca2+ -induced contractions in human hyperplastic prostate; in contrast, nifedipine (10-5 M) almost completely abolished this Ca2+ -induced contraction. These results suggest that tamsulosin had no effect on the voltage-operated calcium channels (VOCC) in human hyperplastic prostate. Additionally, the effect on presynaptic noradrenaline release could also influence the field stimulation-mediated contractions in this tissue. In this study, the data showed that both prazosin (10 nM) and tamsulosin (1 nM) had little effect on the noradrenaline release evoked by electrical field stimulation. In summary, we have demonstrated that tamsulosin is a potent antagonist against field stimulation-induced contractions in human hyperplastic prostate and this antagonistic effect is due mainly to its high affinity for the α1A ?adrenoceptor subtype. We are interested in the drug development in the treatment of BPH. The selective a1-adrenoceptor antagonist is one of the important targets as a substantial body of experimental evidence shows that the contractile properties of human prostate adenoma are mediated primarily by a1-adrenoceptors (Hedlund et al., 1985; Hieble et al., 1985) and a rather dense network of adrenergic nerve fibers has been found within the smooth muscle layer of the prostatic glandular stroma (Vaalasti and Hervonen, 1980). Additionally, endogenous adrenergic stimulation plays an important role in human prostate since the tone of prostatic smooth muscle regulated by the autonomic nervous system is thought to be the dynamic component of bladder outlet obstruction by benign prostatic enlargement (Caine, 1986). We have synthesized some quinazoline-based compounds and found that FH-71, EW-65 and EW-154 are three representatives. The present work demonstrated that FH-71, EW-65 and EW-154 inhibited phenylephrine-induced contractile responses in rat aorta. All of these compounds shifted the concentration-response curves to phenylephrine action in parallel and without diminishing the maximum contraction of the curves suggesting the reversible antagonistic effects on a1-adrenoceptors. Furthermore, at the high concentrations (10 mM) all of these compounds showed no effect on the contractions to high K+ and U46619 revealing that they had little influence on the VOCC and thromboxane A2 receptors. Taken together, the data showed that FH-71, EW-65 and EW-154 were selective a1-adrenoceptor antagonists. Based on functional and binding studies, there exist at least three a1-adrenoceptor subtypes, such as the a1A-, a1B-, and a1D-adrenoceptor subtypes (Ford et al., 1994). Recently, selective a1A-adrenoceptor antagonists have been developed to optimize the therapeutic efficiency of the treatment of benign prostatic hyperplasia, since there is accumulating evidence revealing that a1A-adrenoceptors are the predominant a1-adrenoceptor subtypes in the prostate (Guh et al., 1995; Beduschi et al., 1998). Moreover, the use of a1A-adrenoceptor antagonists could reduce the side effects associated with a-adrenoceptor blockade in other tissues or organs of the body, such as the vascular system (Lepor, 1998). Thus, the development of selective a1A-adrenoceptor antagonists is now an important issue in the management of BPH. To examine the selectivity on a1-adrenoceptor subtypes, rat vas deferens and spleens, respectively for functional determination of a1A- and a1B-adrenoceptor subtypes, were used and the non-selective a1-adrenoceptor antagonist, terazosin, was compared in the present work. The functional data showed that FH-71 exhibited a 19-fold selectivity in a1A- than a1B-adrenoceptor subtypes; whereas, EW-154 showed a 11-fold selectivity in a1B- than a1A-adrenoceptor subtypes. Furthermore, in the functional tension study, we found that FH-71 was the most potent a1-adrenoceptor antagonist among the mentioned compounds in human hyperplastic prostate. It showed 8- and 19-fold potency than that of EW-65 and EW-154, respectively. These results demonstrated the developmental potential of FH-71 in the treatment of benign prostatic hyperplasia. In the present study, we also determined the cytotoxic effects of these compounds in the developmental process. At first, we examined the cytotoxicity using MTT assay method and found that only the high concentration of EW-154 (10 mM) exerted a profound cell death in human prostatic smooth muscle cells. It seems that the cause of prostatic cell death plays a beneficial role in the restriction of prostate size. However, by use of lactate dehydrogenase release assay, EW-154 also induced a significant increase of release reaction suggesting the necrosis other than the apoptosis to EW-154 action. Not only the prostatic cells, EW-154 also induced the cell death in human umbilical vein endothelial cells using the above two assay methods. These results demonstrated that high concentrations of EW-154 might elicit a broad cytotoxic effect in several types of cells. We speculate that the cytotoxic activity of EW-154 could be a result of the guanidine group present in the structure of this compound. In conclusion, we suggest that FH-71 is a selective a1A-adrenoceptor antagonist against muscle contractions in human hyperplastic prostate and possess the potential as a therapeutic agent for clinical symptomatic benign prostatic hyperplasia. In order to define a role of protein kinase C in the regulation of cell growth in hPSMCs, we first examined the influence of protein kinase C inhibitors on serum-induced proliferation in these cells. Staurosporine, a potent protein kinase C inhibitor, caused a significant reduction on serum-induced cell proliferation at nontoxic concentrations (10 and 30 nM) demonstrating the involvement of protein kinase C-dependent mechanism. Additionally, a selective Ca2+-dependent protein kinase C inhibitor, Go-6976 (Martiny-Baron et al., 1993), was employed to elucidate the roles of Ca2+ -dependent protein kinase C isozymes on the regulation of this protein kinase C-signaled cell proliferation. As demonstrated by Martiny-Baron et al. (1993), nanomolar concentrations of Go-6976 inhibited the Ca 2+ -dependent protein kinase C isozymes, whereas even micromolar concentration of this compound had no effect on the kinase activity of the Ca2+-independent protein kinase C isozymes δ, ε, and ξ. Based on a high degree of selectivity, nanomolar concentrations of Go-6976 caused a similar reduction as that did by staurosporine on serum-induced prostatic smooth muscle cell proliferation suggesting that Ca2+-dependent protein kinase C isozymes might be the key enzymes in this protein kinase C-dependent mechanisms. There are accumulating evidences suggest that a variety of protein kinase C isozymes involve in the signal transduction pathway of the proliferative effect in several types of cells (Guizzetti et al., 1996; Huwiler and Pfeilschifter, 1994; Levy et al., 1994). In order to confirm which protein kinase C isozymes involve in human prostatic smooth muscle cell proliferation, we next investigated the expression of protein kinase C isozymes and their translocation following the serum treatment in these cells. Using Western blot technique we determined that hPSMCs expressed protein kinase C-α, -βI, βII, -δ, -ε, and -ξ but did not express protein kinase C-γ, -η, -θ, and -λ. After confirming that hPSMCs express the above six protein kinase C isozymes, we examined the effect of serum on the translocation of these isozymes from a cytosolic to a membrane distribution. The treatment with 10% fetal-calf serum for several time courses caused significant associations of both protein kinase C-βII, and -ε with the membrane fractions in hPSMCs. These results suggest that activation of protein kinase C-βII, and -ε isozymes might be the crucial mechanism in this protein kinase C-mediated pathway. However, in the aforementioned functional experiment, the Ca2+-dependent protein kinase C isozyme (i.e.. Go-6976-sensitive protein kinase C isozyme) might be the key enzyme in this protein kinase C-dependent mechanism demonstrating that growth-promoting effects of serum on hPSMCs are mediated through activation of protein kinase C-βII. Additionally, protein kinase C-ε also showed a marked translocation to the membrane fraction in these cells following serum treatment in this study. However, the role of protein kinase C-e, in mediating cellular functions other than cell proliferation remains further investigation in these cells although it plays a role in the regulation of cell proliferation in some kinds of cells (Gomez et al., 1995). The involvement of protein kinase C-βII in mediating hPSMC proliferation was supported by the observation that prolonged treatment with high concentrations of phorbol 12-myristate 13-acetate for 20 h, which downregulates most protein kinase C isozymes, completely eliminated protein kinase C-βII in these cells; furthermore, in parallel experiments, prolonged exposure (20 h) to high concentrations of phorbol 12-myristate 13-acetate also significantly reduced serum-stimulated cell proliferation (31.4 + 3.5% reduction as compared with the serum treatment control) in these cells. In summary, we demonstrated that the proliferative effects of serum on hPSMCs involve the activation of protein kinase C, also characterized the expression of various protein kinase C isozymes in these cells. The association of protein kinase C-βII with the membrane fraction significantly increased during serum-induced cell proliferation. These findings suggested that protein kinase C-βII may play a crucial role in serum-evoked proliferative signaling in hPSMCs. Further studies are required to determine the exact role of protein kinase C-ε during serum-induced functional expression in these cells. It has been suggested that circulating endogenous inhibitors of the plasma Na+ pump (Na+-K+ ATPase) could be responsible for increased vascular smooth muscle tone in some forms of hypertension (Masugi, 1986; Hamlyn, 1982). In addition to vascular smooth muscle, the activity of Na+-K+ ATPase is a crucial factor in the regulation of several cellular functions, including the contractility of smooth muscles, the release of neurotransmitter, and the hypertrophic and proliferative effects of several types of cells (Guh, 1995; Guh, 2000; Murata, 1996). In this study, we have examined the effect of ouabain on the modulation of hPSMC growth, the predominant factor of BOO in patients with BPH. The hPSMCs were used as the experimental model, since smooth muscle represents about 40% of the area density of BPH tissues (Shapiro, 1992). At first, we have examined the effect of ouabain on the growth regulation in hPSMCs using MTT assay method other than radioactive analysis because of the prevention of isotope problem; furthermore, this assay method has been used for the measurement of both cell proliferation and cytotoxic effect on several types of cells and provides reproducible and accurate measurements of cell killing and proliferation compared with the [3H]thymidine incorporation assay and trypan blue exclusion test. Interestingly, ouabain stimulated the cell proliferation in very low concentrations (0.01 to 1 nM), which is also the therapeutic level of cardiac glycoside; whereas induced significant cell death in concentrations more than ten nanomolar levels. In view of the proliferative action to ouabain, only 10 to 30% increase of cell number was observed. However, this modest increase of cell number might cause a profound clinical syndrome in patients with BPH, since there are many clinical analyses revealing that the small increase in prostate size significantly deteriorates syndrome in men with BPH and, similarly, the small reduction in prostate volume (about 30%) profoundly improves their symptoms (McConnell, J.D, 1998; Jonler, 1994). Of note, this proliferative action to ouabain was exhibited in the subnanomolar concentrations, the therapeutic range in patients with the administration of cardiac glycosides, indicating the possible involvement of BPH pathophysiology in a clinical setting. The proliferative effect of ouabain was also observed in some other types of cells (Balk, 1984). Among these studies, Ca2+ influx or mobilization caused by ouabain is a key event in the initiation of cell replication. Therefore, further investigation regarding the signal pathways initiated by ouabain was carried out on the roles of Ca2+ and calmodulin in this study. The results demonstrated that membrane permeable Ca2+/calmodulin inhibitor W-7 significantly diminished the proliferative action to ouabain suggesting the Ca2+ mobilization is necessary for ouabain action. In addition, we also examined the involvement of p42/44 MAPK isoforms in this study, as it is suggested that the activation of p42/44 MAPKs plays a crucial role in the regulation of cell growth (Seger, 1995). In the present study, ouabain induced the activation and phosphorylation of p42/44 MAPKs in a concentration-dependent manner suggesting the involvement of p42/44 MAPK pathway in ouabain-induced hPSMC proliferation. However, this effect has been declined after the exposure of cells to cytotoxic concentration of ouabain. The inhibition of p42/44 MAPK could stimulate stress (osmotic or heat shock)-induced apoptosis in SKT6 cells (Nagata et al, 1999); in contrast, activation of p42/44 MAPKs suppressed stress-induced apoptosis. It thus appears that the activation of p42/44 MAPKs is not only contributed to the proliferative action but also necessary for the survival in hPSMCs and some other types of cells. In our unpublished data, we have also examined the effect of Ca2+-free/EGTA medium on ouabain-induced activation of p42/44 MAPKs, and the results showed the disappearance of ouabain-induced effect confirming the requirement of extracellular Ca2+ and/or Ca2+ mobilization on ouabain action. However, among this experiment Ca2+-free/EGTA medium alone induced a marked increase of p42/44 MAPK phosphorylation. This effect was also observed in rat liver epithelial cells by Maloney et al (1999). They have suggested that the depletion of intracellular Ca2+ with EGTA caused inactivation of protein phosphatase 2A and protein tyrosine phosphatases. Whether this effect to Ca2+ deprivation was due to the suppression of protein phosphatases remained further investigation in hPSMCs. Scientific interest has focused on the reduced cell death theory in prostate and it has been suggested that an imbalance between programmed cell death (apoptosis) and cell proliferation may result in the development of BPH (Burnett, 1995). In this study, we have examined the effect of ouabain on the apoptotic response using TUNEL-reaction technique to identify apoptotic cells. The results showed that ouabain could induce the profound cell apoptosis in high concentrations. Although the cytotoxic concentration of ouabain more than ten times of therapeutic range, it is important to define the apoptotic action to ouabain for the understanding of apoptotic mechanism in hPSMCs. Apoptosis, a morphological distinct form of programmed cell death, requires the participation of endogenous cellular enzymes. Central to the apoptotic program is a family of cysteine proteases termed caspases. It appears that apoptotic processes stimulated by a variety of stimuli converge on the activation of a member of the caspase family. In living cells caspases are present as inactive zymogens and become activated following the apoptotic stimuli. To date, more than ten distinct human caspase genes have been identified (Villa, 1997). Among these caspases, the activation of caspase-3 is the crucial event in a variety of cells, which leads to the execution of apoptosis. In this study, DEVD-CHO, a specific caspase-3 inhibitor, suppressed the ouabain-induced apoptosis in a concentration-dependent manner. Furthermore, the apoptotic action to ouabain correlated with the increase of caspase-3 activity using an enzymatic assay method. From these results we suggest that caspase-3 is the key executioner of ouabain-induced apoptosis in hPSMCs. To extend these finding to other apoptotic stimulus, hPSMCs were exposed to cytotoxic concentrations of staurosporine (10-7 to 3x10-6 M) and similar results were obtained as for ouabain-treated cells (unpublished data). It thus appears that caspase-3 activation is a general marker of apoptosis in hPSMCs. In the present study, high concentrations of ouabain also induced necrotic cell death as measured by increased LDH release. Such two fundamental forms of cell death, that is the apoptosis and necrosis, occurred after the apoptotic stimulus were also observed in a variety of cell types. In the report by Filipovic et al.(1999) they have suggested that oxidant-induced apoptosis activates poly(ADP-ribose) polymerase (PARP) and that the subsequent ATP and NAD depletion contribute to necrotic cell death in renal epithelial cells. In the report by Li et al. (1999) they observe that b-lapachone induced cell death in a spectrum of human carcinoma cells. It induced apoptosis in human ovary, colon, and lung cancer cells, and necrotic cell death in four human breast cancer cell lines; moreover, mitochondrial cytochrome C release was found in both apoptosis and necrosis. They have suggested that the release of cytochrome C may be the shared upstream event in apoptotic and necrotic cell death. Interestingly, in our unpublished study, staurosporine induced profound apoptotic and necrotic cell death in rat prostatic stromal cells. Both forms of cell death were preceded by a rapid release of cytochrome C as a shared mechanism. However, is there any common mechanism in apoptotic and necrotic death of hPSMCs needs detailed investigation in our further study. In summary, in the present study we demonstrate that ouabain at different concentrations causes dual effects on the proliferation and apoptosis in hPSMCs. At lower concentrations, ouabain promotes hPSMC proliferation via a Ca2+-dependent mechanism and activation of MEK-p42/44 MAPK pathway; whereas it induces cell apoptosis via activation of caspase-3 activity at higher concentrations. The necrotic cell death also accounts for this ouabain action