Rac may be a link connecting the control of prostate smooth muscle tone with proliferation of smooth muscle cells. Improvements in LUTS suggestive of BPH by Rac inhibitors appears possible.

Prostate smooth muscle tone and hyperplastic growth are involved in the pathophysiology and treatment of male lower urinary tract symptoms (LUTS). Available drugs are characterized by limited efficacy. Patients’ adherence is particularly low to combination therapies of 5α-reductase inhibitors and α1-adrenoceptor antagonists, which are supposed to target contraction and growth simultaneously. Consequently, molecular etiology of benign prostatic hyperplasia (BPH) and new compounds interfering with smooth muscle contraction or growth in the prostate are of high interest. Here, we studied effects of p21-activated kinase (PAK) inhibitors (FRAX486, IPA3) in hyperplastic human prostate tissues, and in stromal cells (WPMY-1). In hyperplastic prostate tissues, PAK1, -2, -4, and -6 may be constitutively expressed in catecholaminergic neurons, while PAK1 was detected in smooth muscle and WPMY-1 cells. Neurogenic contractions of prostate strips by electric field stimulation were significantly inhibited by high concentrations of FRAX486 (30 μM) or IPA3 (300 μM), while noradrenaline- and phenylephrine-induced contractions were not affected. FRAX486 (30 μM) inhibited endothelin-1- and -2-induced contractions. In WPMY-1 cells, FRAX486 or IPA3 (24 h) induced concentration-dependent (1–10 μM) degeneration of actin filaments. This was paralleled by attenuation of proliferation rate, being observed from 1 to 10 μM FRAX486 or IPA3. Cytotoxicity of FRAX486 and IPA3 in WPMY-1 cells was time- and concentration-dependent. Stimulation of WPMY-1 cells with endothelin-1 or dihydrotestosterone, but not noradrenaline induced PAK phosphorylation, indicating PAK activation by endothelin-1. Thus, PAK inhibitors may inhibit neurogenic and endothelin-induced smooth muscle contractions in the hyperplastic human prostate, and growth of stromal cells. Targeting prostate smooth muscle contraction and stromal growth at once by a single compound is principally possible, at least under experimental conditions.

Contractions following concomitant confrontation of human prostate tissue with noradrenaline and endothelin-1 are not additive. Endothelin-1 is sufficient to induce a smooth muscle tone resembling that of noradrenaline. This may replace lacking α -adrenergic tone under therapy with α -blockers, explaining the limited efficacy of α -blockers in LUTS treatment. Contractions by thromboxane and endothelin-1 may be additive, and may exceed α -adrenergic tone. Prostate 77:697-707, 2017. © 2017 Wiley Periodicals, Inc.

Edited by Henrik G. Dohlman Voiding symptoms in benign prostatic hyperplasia (BPH) are driven by prostate smooth muscle contraction and prostate growth. Smooth muscle contraction in the prostate and otherorganscriticallydependsonactivationofthesmallmonomeric GTPase RhoA and probably Rac1. A role of another GTPase, ADP-ribosylation factor 6 (ARF6), for smooth muscle contraction has been recently suggested by indirect evidence but remains to be proven for any organ. Here, we report effects of NAV2729, an inhibitor with assumed specificity for ARF6, in human prostate tissues and cultured prostate stromal cells (WPMY-1). NAV2729 (5 M) inhibited neurogenic and ␣ 1-adrenergic contractions of human prostate tissues. Contractions induced by endothelin-1, by the thromboxane A 2 agonist U46619, or by high molar KCl were not inhibited. Correlation analyses suggested up-regulation of prostatic ARF6 expression with increasing degree of BPH, as ARF6 expression increased with the content of prostate-specific antigen (PSA) of prostate tissues. NAV2729 inhibited ARF6 activity but not other GTPases (ARF1, RhoA, Rac1) in prostate tissues and in WPMY-1 cells. Proliferation of WPMY-1 cells was inhibited concentration-dependently by NAV2726, as reflected by decreased viability, 5-ethynyl-2-deoxyuridine (EdU) assay, colony formation assay, and expression of Ki-67. Silencing of ARF6 expression mimicked effects of NAV2729 on viability and in the EdU assay. Effects of NAV2729 on viability and proliferation were attenuated in cells with silenced ARF6 expression. Our findings suggest that a NAV2729-sensitive mechanism promotes adrenergic contraction and stromal cell growth in the human prostate, which is probably ARF6-mediated. Similar actions in other organs and urodynamic effects of NAV2729 appear possible.

BACKGROUND AND PURPOSEIn men with benign prostatic hyperplasia, increased smooth muscle tone in the prostate may lead to bladder outlet obstruction and subsequent lower urinary tract symptoms. Consequently, medical treatment aims to inhibit prostate smooth muscle contraction. However, the efficacy of the treatment options available is limited, and improved understanding of mechanisms of prostate smooth muscle contraction and identification of new targets for medical intervention are mandatory. Several studies suggest that LIM kinases (LIMKs) promote smooth muscle contraction; however, this has not yet been examined. Here, we studied effects of the LIMK inhibitors on prostate smooth muscle contraction. EXPERIMENTAL APPROACHHuman prostate tissues were obtained from radical prostatectomy. Phosphorylation of cofilin, a LIMK substrate, was examined using a phospho-specific antibody. Smooth muscle contractions were studied in organ bath experiments. KEY RESULTSReal-time PCR, Western blot and immunofluorescence suggested LIMKs are expressed in smooth muscle cells of prostate tissues. Two different LIMK inhibitors, SR7826 (1 μM) and LIMKi3 (1 μM), inhibited contractions of prostate strips, which were induced by electrical field stimulation, α 1 -adrenoceptor agonists phenylephrine and methoxamine and the TXA 2 analogue, U46619. LIMK inhibition in prostate tissues and cultured stromal cells (WPMY-1) was confirmed by cofilin phosphorylation, which was reduced by SR7826 and LIMKi3. In WPMY-1 cells, SR7826 and LIMKi3 caused breakdown of actin filaments and reduced viability. CONCLUSIONS AND IMPLICATIONSSmooth muscle tone in the hyperplastic human prostate may underlie the effects of LIMKs, which promote contraction. Contraction of prostate strips can be inhibited by small molecule LIMK inhibitors.

Background and Purpose In benign prostatic hyperplasia, increased prostate smooth muscle tone and prostate volume may contribute alone or together to urethral obstruction and voiding symptoms. Consequently, it is assumed there is a connection between smooth muscle tone and growth in the prostate, but any molecular basis for this is poorly understood. Here, we examined effects of Src family kinase (SFK) inhibitors on prostate contraction and growth of stromal cells. Experimental Approach SFK inhibitors, AZM475271 and PP2, were applied to human prostate tissues to assess effects on smooth muscle contraction, and to cultured stromal (WPMY‐1) and c‐Src‐deficient cells to examine effects on proliferation, actin organization and viability. Key Results SFKs were detected by real time PCR, western blot and immunofluorescence in human prostate tissues, some being located to smooth muscle cells. AZM475271 (10 μM) and PP2 (10 μM) inhibited SFK in prostate tissues and WPMY‐1 cells. Both inhibitors reduced α1‐adrenoceptor‐mediated and neurogenic contraction of prostate strips. This may result from cytoskeletal deorganization, which was observed in response to AZM475271 and PP2 in WPMY‐1 cells by staining of actin filaments with phalloidin. This was paralleled by reduced proliferation of wildtype but not of c‐Src‐deficient cells; cytotoxicity was mainly observed at higher concentrations (>50 μM). Conclusions and Implications In human prostate, smooth muscle tone and growth are both controlled by an SFK‐dependent process, which may explain their common role in bladder outlet obstruction. Targeting prostate smooth muscle tone and prostate growth simultaneously by a single compound may, in principal, be possible.

No abstract

Non-adrenergic prostate smooth muscle contractions may account for the limited effectiveness of α1-adrenoceptor antagonists, which are the first-line option for medical treatment of voiding symptoms suggestive of benign prostatic hyperplasia. In non-human prostates, purinergic agonists induce contractions reaching similar magnitudes as α1-adrenergic contractions. However, evidence for the human prostate is highly limited, and pointed to much weaker purinergic contractions. Here, we examined contractions of different purinergic agonists in human prostate tissues. Tissues were obtained from radical prostatectomy. Contractions were studied in an organ bath, and expression of purinergic receptors was studied by RT-PCR. Electric field stimulation (EFS)–induced contractions amounted to 104% of KCl-induced contractions (95% CI: 84–124%). From all tested agonists, only ATP induced concentration-dependent contractions, reaching an average maximum of 18% (12–24%) of KCl. Maximum tensions following application of other agonists averaged to 7.1% of KCl for α,β-methylene-ATP (1.8–12.4%), 3.9% for β,γ-methylene-ATP (2.0–5.4%), 3.1% for 2-methylthio-ATP (− 0.1–6.3%), and 5.1% for ATPγS (1.0–9.2%). Responses were not affected by the P2X antagonist NF023 or the P2Y antagonist PPADS. mRNA expression of P2X1-4 correlated with expression of a marker for catecholaminergic nerves, although neither ATP, NF023, nor PPADS changed EFS-induced contractions. Correlation between expression of receptors and the smooth muscle marker calponin was not observed. Our findings point to a low relevance of purinergic contractions in the human prostate, compared to other contractile stimuli in the human prostate and compared to purinergic contractions in non-human prostates. Purinergic contractions in the human prostate are not sensitive to NF023 or PPADS.