Inhibition of human prostate smooth muscle contraction by the inhibitors of protein kinase C, GF109203X, and Go6983.

INTRODUCTION: Prostate smooth muscle contraction is promoted by receptor-induced activation of intracellular signaling pathways. The presumed involvement in etiology and medical treatment of lower urinary tract symptoms (LUTS) suggestive of benign prostatic hyperplasia (BPH) imparts a high clinical relevance to prostate smooth muscle contraction, which is contrasted by incomplete understanding at the molecular level. Involvement of protein kinase C (PKC) has been commonly assumed, but available studies were limited to nonhuman prostate smooth muscle or cell cultures. Here, we examined the effects of the PKC inhibitors Go6983 and GF109203x on contractions of human prostate tissues. METHODS: Prostate tissues were obtained from radical prostatectomy. Contractions were induced by electric field stimulation (EFS), α1 -adrenergic agonists (noradrenaline, phenylephrine, methoxamine), thromboxane A2 analog U46619, endothelin-1, or calcium chloride in an organ bath. RESULTS: GF109203X (500 nM) and Go6983 (300 nM) reduced EFS-, noradrenaline-, phenylephrine-, methoxamine-, and U46619-induced contractions of human prostate tissues, with maximum inhibitions approaching up to 55%. Using concentrations of 3 µM, GF109203X and Go6983 inhibited EFS- and noradrenaline-induced contractions, with similar effect sizes as 500 and 300 nM, respectively. Endothelin-1-induced contractions were not inhibited by GF109203X, and to neglectable extent by Go6983. After depolarization in calcium-free solution, calcium chloride-induced concentration-dependent contractions, which were inhibited by GF109203X and Go6983. CONCLUSIONS: GF109203X and Go6983 inhibit neurogenic, α1 -adrenergic, and thromboxane A2 -induced smooth muscle contractions in the human prostate, suggesting a role of PKC for human prostate smooth muscle contraction. The inhibition may by be imparted by inhibition of calcium sensitivity.

Inhibition of Full Smooth Muscle Contraction in Isolated Human Detrusor Tissues by Mirabegron Is Limited to Off-Target Inhibition of Neurogenic Contractions.

Mirabegron is used for treatment of storage symptoms in overactive bladder (OAB) caused by spontaneous bladder smooth muscle contractions. However, owing to limitations in available studies using human tissues, central questions are still unresolved, including mechanisms underlying improvements by mirabegron and its anticontractile effects in the detrusor. Here, we assessed concentration-dependent mirabegron effects on contractions of human detrusor tissues in frequency-response curves and concentration-response curves for different cholinergic and noncholinergic agonists. Detrusor tissues were sampled from patients undergoing radical cystectomy. Contractions were induced by electric field stimulation (EFS) and by cumulative concentrations of cholinergic agonists, endothelin-1, and the thromboxane A2 analog U46619. EFS-induced contractions were inhibited using 10 µM mirabegron, but not using 1 µM. Inhibition by 10 µM mirabegron was resistant to the ß 3-adrenergic antagonist L-748,337. Concentration-dependent contractions by carbachol were not inhibited by 1 µM or 10 µM mirabegron. Concentration-response curves for methacholine were slightly right-shifted by 10 µM, but not 1 µM mirabegron. Concentration-dependent contractions by endothelin-1 or U46619 were not changed by mirabegron. In contrast, the muscarinic antagonist tolterodine right-shifted concentration-response curves for carbachol and methacholine and inhibited EFS-induced contractions. In conclusion, inhibition of neurogenic contractions in isolated detrusor tissues by mirabegron requires concentrations highly exceeding known plasma levels during standard dosing and the known binding constant (Ki values) for ß 3-adrenoceptors. Full contractions by cholinergic agonists, endothelin-1, and U46619 are not affected by therapeutic concentrations of mirabegron. Improvements of storage symptoms are most likely not imparted by inhibition of ß 3-adrenoceptors in the bladder wall itself. SIGNIFICANCE STATEMENT: Mirabegron is used for overactive bladder (OAB) treatment, but the underlying mechanisms are unclear, and preclinical and clinical findings are controversial due to limitations in available studies. Our findings suggest that inhibition of detrusor contractions by mirabegron is limited to neurogenic contractions, which requires unphysiologic concentrations and does not involve ß 3-adrenoceptors. Mechanisms accounting for improvements of OAB by mirabegron are located outside the urinary bladder.

Clinical Evaluation of Single-Use, Fiber-Optic, and Digital Ureterorenoscopes in the Treatment of Kidney Stones.

INTRODUCTION: Indication of ureteroscopy for the treatment of urolithiasis has expanded immensely over the last decade. Fiber-optic and digital reusable instruments present the standard in clinical practice, but various newly available single-use devices might offer an exciting alternative. To date, the evidence is limited to clinical evaluation and efficacy of single-use ureteroscopes (URS) compared to standard instruments. Therefore, we evaluate a single-use instrument's clinical characteristics and efficacy in direct comparison with a fiber-optic and digital device. METHODS: A prospective study was conducted for patients undergoing endoscopic therapy for urolithiasis at a tertiary care center. We evaluated the different instruments' clinical performance in categories of visibility, the stability of visibility, irrigation flow, and surgeon's satisfaction. Statistical analyses were performed by SPSS using the Chi-Quadrat and Kruskal-Wallis test. A p value of p ≤ 0.05 was defined as statistically significant. RESULTS: A total number of 77 patients were included and distributed as follows: 35 (46.7%) single-use, 19 (25.3%) digital, and 23 (28%) fiber-optic URS. Patients' characteristics were homogenous over the three cohorts in sex, stone amount, and localization. The stone-free rate was equal in all three cohorts (p = 0.31). We identify stability of visibility, irrigation flow, and satisfaction were equal in all cohorts (p = 0.73; p = 0.20; p = 0.20). We report a significant difference in visibility, with 100% rated excellent in the digital URS group (p = 0.028). DISCUSSION/CONCLUSIONS: Single-use URS achieve comparable clinical outcomes with equal stone-free rates in direct comparison with fiber-optic and digital reusable instruments. Accordingly, single-use devices present an adequate alternative for endoscopic therapy of urolithiasis.

Evaluation of intrarenal pressure stability at different lithotripter suction settings in a porcine kidney model.

PURPOSE: Using the Swiss LithoClast® Trilogy, urinary stones can be fragmented and removed simultaneously by suction at different selectable suction settings. The aim was to evaluate pressure stability at different settings and test stone fragmentation and suction at the optimal settings. METHODS: In an ex vivo porcine kidney model, we recorded intrarenal pressure levels with different suction levels. Storz® Nephroscopes MIP-M and MIP-L and Swiss LithoClast® Trilogy probes were used. RESULTS: Pressure stabilized at 19 cm H2O with the MIP-M at 1 m gravity irrigation with no instrument introduced. After inserting the 1.5 mm probe, the pressure dropped to 5 cm H2O. With a suction setting of 10%, the pressure stabilized at 3 cm H2O and remained stable for the maximum time of 120 s. After increasing the suction to 20, 30, 40, and 50%, we recorded the pressure drop time to 0 after 22, 14, 11, and 8 s. Using the MIP-L, pressure stabilized at 44 cm H2O and decreased to 8 cm H2O after inserting the 3.4 mm probe. With 10% suction, a pressure stabilization was measured at 2 cm H2O and remained stable for 120 s. At suction levels of 20 and 30%, the pressure drop time to 0 was 6 and 5 s. With a 10% suction, removing stones was efficient, and the kidney's filling volume was maintained. CONCLUSIONS: When using the LithoClast® Trilogy, a suction setting of 10% seems to be optimal for the treatment of urinary calculi when applying suction continuously.

Adherence to guidelines in the management of urolithiasis: are there differences among distinct patient care settings?

INTRODUCTION: Urolithiasis is a common diagnosis in urology. New technologies offer a variety of diagnostic and therapy and consequently display a financial burden on healthcare systems. Hence, clinical practice guidelines (CPG) are essential to implement evidence-based medicine and assure a standard of care considering limited resources. To date, there is no evidence of the use and adherence to CPG on urolithiasis. MATERIAL AND METHODS: Therefore, we performed a cross-sectional study to analyze the use of CPG on urolithiasis. Data collection was carried out by a questionnaire given to 400 German urologists. The survey included use and adherence to guidelines, evaluation of the clinical situation, therapy spectrum, and workplace. In total, 150 (37%) questionnaires were received and included in our survey. Statistics were performed by SPSS using Chi-quadrat test/Fisher's exact test. RESULTS: In our study, urologists were office based, hospital affiliated, non-academic, or academic centers in 53%, 32%, 16% and 5%, respectively. In 74% and 70%, urologists adhere to CPG in diagnostic and therapy. Interestingly, workplace and therapy spectrum determines the use of different CPG (p = 0.01; p = 0.022). Academic urologists were more likely to use international CPG of EAU (40%), while outpatient urologists significantly orientated on national CPG (46%). 86% of urologists with high volume of urolithiasis practice interventions in contrast to 53% in low volume (p = 0.001). More than 80% of urologists use short versions and app version of CPG. CONCLUSION: We firstly describe compliance and the use of CPG on urolithiasis. EAU and DGU present the most commonly used CPG. Short version and app version of CPG find frequent clinical utilization.

Comparison of stone elimination capacity and drilling speed of endoscopic clearance lithotripsy devices.

PURPOSE: To investigate the fragmentation capacity, clearance time, and drilling speed of combined ultrasonic with impact dual-energy and single energy ultrasonic lithotripter devices. METHODS: Stone fragmentation and clearance tests were performed under direct view in an underwater layered hemisphere by four different operators using artificial stones (n = 10/operator). Time for complete clearance was measured. Drilling tests were performed using an underwater setup, consisting of a mounting rack for fixing the lithotripter handpiece with the probe in vertical position and in contact with the stone phantom placed on one side of a balance for defined and constant contact application pressure equivalent to 450 g load. Time until complete perforation or in case of no perforation, the penetration depth after 60 s into the stone sample was recorded. Four devices, one single energy device (SED), one dual-energy dual probe (DEDP), two dual-energy single probe (DESP-1, DESP-2), with different parameters were tested. RESULTS: Stone fragmentation and clearance speed were significantly faster for dual-energy device DESP-1 compared to all other devices (p < 0.001). Using DESP-1, the clearance time needed was 26.0 ± 5.0 s followed by DESP-2, SED and DEDP requiring 38.4 ± 5.8 s, 40.1 ± 6.3 s and 46.3 ± 11.6 s, respectively. Regarding the drilling speed, DESP-1 was faster compared to all other devices used (p < 0.05). While the drilling speed of DESP-1 was 0.69 ± 0.19 mm/s, compared to 0.49 ± 0.18 mm/s of DESP-2, 0.47 ± 0.09 mm/s of DEDP, and 0.19 ± 0.03 mm/s of SED. CONCLUSIONS: The dual-energy/single-probe device combining ultrasonic vibrations with electromechanical impact was significantly faster in fragmentation and clearing stone phantoms as well as in drilling speed compared to all other devices.

Impact of previous transurethral prostate surgery on health-related quality of life after radical prostatectomy: Does the interval between surgeries matter?

PURPOSE: To assess the impact of previous transurethral surgery for benign prostate enlargement (BPE) and time interval between procedures on functional outcomes and health-related quality of life (HRQOL) after radical prostatectomy (RP). METHODS: A propensity score-matched patient cohort [n = 685, (513 without previous BPE surgery, 172 with BPE surgery)] was created and HRQOL was pre- and postoperatively assessed using validated questionnaires (EORTC QLQ-C30). Urinary continence was measured via ICIQ-SF questionnaire and pad usage. Multivariable analysis included binary logistic and Cox regression models (p < 0.05). RESULTS: Median follow-up was 18 months. There was no significant difference in recurrence-free survival in multivariate analysis (HR 0.66, 95%CI 0.40-1.07, p = 0.093). We observe higher mean ICIQ-SF scores (5.7 vs. 8.2, p < 0.001) and daily pad usage (1.3 vs. 2.5, p < 0.001), and decreased continence recovery (OR 0.46, 95%CI 0.30-0.71, p < 0.001) for patients with BPE surgery. Postoperative general HRQOL scores were significantly lower for patients with previous BPE surgery (70.6 vs. 63.4, p = 0.003). In multivariate analysis, continence recovery (OR 5.19, 95%CI 3.10-8.68, p < 0.001) but not previous BPE surgery (0.94, 0.57-1.54, p = 0.806) could be identified as independent predictors of good general HRQOL. There was no significant correlation between time interval between both surgeries and continence (p = 0.408), and HRQOL (p = 0.386) outcomes. CONCLUSIONS: We observe favourable continence outcomes for patients without previous BPE surgery. Our results indicate that RP can be safely performed after transurethral BPE surgery, regardless of the time interval between both interventions.

The transient receptor potential A1 ion channel (TRPA1) modifies in vivo autonomous ureter peristalsis in rats.

AIMS: The current study aimed to explore the expression of transient receptor potential A1 ion channels (TRPA1) in the rat ureter and to assess if TRPA1-active compounds modulate ureter function. METHODS: The expression of TRPA1 in rat ureter tissue was studied by immunofluorescence. The TRPA1 distribution was compared to calcitonin gene-related peptide (CGRP), α-actin (SMA1), anoctamin-1 (ANO1), and c-kit. For in vivo analyses, a catheter was implanted in the right ureter of 50 rats. Ureter peristalsis and pressures were continuously recorded by a data acquisition set-up during intraluminal infusion of saline (baseline), saline plus protamine sulfate (PS; to disrupt the urothelium), saline plus PS with hydrogen sulfide (NaHS) or cinnamaldehyde (CA). Comparisons were made between rats treated systemically with vehicle or a TRPA1-antagonist (HC030031). RESULTS: TRPA1-immunoreactive nerves co-expressed CGRP and were mainly located in the suburothelial region of the ureter. Immunoreactivity for TRPA1 was also encountered in c-kit-positive but ANO1-negative cells of the ureter suburothelium and wall. In vivo, HC030031-treated rats had elevated baseline peristaltic frequency (p < 0.05) and higher intraluminal pressures (p < 0.01). PS increased the frequency of ureter peristalsis versus baseline in vehicle-treated rats (p < 0.001) but not in HC030031-treated rats. CA (p < 0.001) and NaHS (p < 0.001) decreased ureter peristalsis. This was counteracted by HC030031 (p < 0.05 and p < 0.01). CONCLUSIONS: In rats, TRPA1 is expressed on cellular structures considered of importance for peristaltic and mechanoafferent functions of the ureter. Functional data indicate that TRPA1-mediated signals regulate ureter peristalsis. This effect was pronounced after mucosal disruption and suggests a role for TRPA1 in ureter pathologies involving urothelial damage.

A NAV2729-sensitive mechanism promotes adrenergic smooth muscle contraction and growth of stromal cells in the human prostate.

Voiding symptoms in benign prostatic hyperplasia (BPH) are driven by prostate smooth muscle contraction and prostate growth. Smooth muscle contraction in the prostate and other organs critically depends on activation of the small monomeric 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 µm) inhibited neurogenic and α1-adrenergic contractions of human prostate tissues. Contractions induced by endothelin-1, by the thromboxane A2 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.

Comparative evaluation of tissue damage induced by ultrasound and impact dual-mode endoscopic lithotripsy versus conventional single-mode ultrasound lithotripsy.

OBJECTIVE: The aim of our study was to perform comparative investigation of the tissue safety of three different endoscopic lithotripter devices including a new single-probe/dual-energy lithotripter in an in vivo animal model. The Swiss LithoClast Trilogy was compared to the Storz Calcuson and the Swiss LithoClast Vario. The safety test simulated the accidental direct contact between lithotripter probes and the urothelium, which can occur when sliding off a stone or drilling through a calculus during lithotripsy. The safety test included a smallest (1.5 mm) and largest (3.3/3.4 mm) probe diameter per device. METHODS: Testing was performed in nine pigs (three animals per device). The bladder tissue was exposed to direct lithotripter probe contact at maximum power for 10 s to produce visible tissue lesions. Acute tissue trauma was evaluated using a simplified scoring model describing the expected bladder wall injuries for histological examination. After 7 days, all animals were killed, necropsied and examined post mortem. For between-group comparisons regarding microscopic histopathologic features, a Chi-square test was used. A p value < 0.05 was considered to be statistically significant. RESULTS: Irrespective of the lithotripter used, no systemic signs of toxicity were observed. Histologically, signs of normal ongoing healing were observed on the bladder mucosa. There were no significant differences in histological findings taking changes of the epithelium (p = 0.360), the leucocyte infiltration (p = 0.123), the vascular congestion (p = 0.929) and the edema (p = 1.0) between the groups into account. CONCLUSIONS: The results of this study demonstrated a comparable safety between all lithotripsy devices.

Health-related quality of life after open and robot-assisted radical prostatectomy in low- and intermediate-risk prostate cancer patients: a propensity score-matched analysis.

PURPOSE: Introduction of robot-assisted radical prostatectomy (RARP) has revolutionized the therapeutic landscape of organ-confined prostate cancer (PCa). However, comparative analyses focused on health-related quality of life (HRQOL) after RARP and open retropubic prostatectomy (ORP) are sparse. METHODS: In the current retrospective analysis, inclusion criteria encompassed PSA ≤ 10 ng/ml, ≤ pT2c, ISUP ≤ 3, age ≤ 65 years, and preoperative continence. A propensity score-matched patient cohort [n = 418 (ORP: 209, RARP: 209)] was created and HRQOL was prospectively assessed based on validated questionnaires (EORTC QLQ-C30) preoperatively, 3 months, 12 months, and 24 months postoperatively. Primary endpoint was good general HRQOL based on previously published cut-off values. Erectile function was measured via IIEF-5, urinary continence via ICIQ-SF questionnaire. Multivariable analysis included binary logistic regression models (p < 0.05). RESULTS: Open retropubic prostatectomy and RARP cohorts were well balanced. General HRQOL was significantly higher for ORP compared to RARP after 3 months (70.1 vs. 61.6, p = 0.001), but not at the remaining follow-up time points. There were no significant differences for the remaining QLQ-C30 functioning and symptom scores. In multivariable analysis stratified for IIEF-5 and ICIQ-SF scores and surgeon experience, RARP could be confirmed as a marginally independent predictor for lower ratios of good general HRQOL after 3 months (OR 0.464, 95% CI 0.215-0.999; p = 0.050) without any differences at the remaining time points. CONCLUSIONS: The current study addresses various HRQOL outcomes over a postoperative period of up to 2 years in a homogenous propensity score-matched contemporary cohort. Marginally better general HRQOL outcomes could be detected for ORP compared to RARP 3 months postoperatively.

New strategies for inhibition of non-adrenergic prostate smooth muscle contraction by pharmacologic intervention.

BACKGROUND: Inhibition of prostate smooth muscle contraction by α1 -adrenoceptor antagonists (α1 -blockers) is a first-line medical treatment of lower urinary tract symptoms suggestive of benign prostatic hyperplasia. Increased smooth muscle tone in the hyperplastic prostate may drive urethral obstruction, resulting in bladder outlet obstruction and voiding symptoms. However, efficacy of α1 -blockers is limited, as non-adrenergic mediators including endothelin-1 and thromboxane A2 (TXA2 ) increase prostate smooth muscle tension in parallel to α1 -adrenoceptors. This may maintain urethral obstruction despite therapy with α1 -blockers. Consequently, future treatment options with higher efficacy need to target α1 -adrenergic and non-adrenergic contractions simultaneouly. Recently, several compounds were reported to inhibit adrenergic or neurogenic prostate contractions, however, their effects on non-adrenergic contraction are unknown. Here, we examined effects of inhibitors for Rac-GTPase, Src family kinases (SFKs), and p21-activated kinases (PAKs) on non-adrenergic prostate contractions. METHODS: Prostate tissues were obtained from radical prostatectomy. Contractions were studied in an organ bath. Viability of cultured stromal cells was assessed by CCK-8 assay. RESULTS: Inhibition of α1 -adrenergic contractions by Rac inhibitors EHT1864 (100 µM) and NSC23766 (100 µM), and SFK inhibitors AZM475721 (10 µM) and PP2 (10 µM) was confirmed by inhibition of methoxamine-induced contractions. No effects of the PAK inhibitors FRAX486 (30 µM) and IPA3 (300 µM) on α1 -adrenergic contraction were confirmed by absent effects on methoxamine-inuced contractions. EHT1864 caused inhibition of endothelin-1- and U46619-induced contractions. EHT1864 reduced the viability of stromal cells concentration- and time-dependently. EHT1864 attenuated KCl-induced contractions of prostate strips only slightly, so that toxic effects may not account alone for inhibition of agonist-induced contractions. NSC23766 inhibited U46619-induced contractions, but not endothelin-1-induced contractions. AZM475271 had no effects on endothelin-1- or U46619-induced contractions, while PP2 inhibited U46619- but not endothelin-1-induced contractions. FRAX486 caused inhibition of U46619-induced contractions. IPA3 inhibited U46619-, but not endothelin-1-induced contractions. CONCLUSIONS: Of all six inhibitors, EHT1864 seems to be most promising from a translational point of view, as it inhibited TXA2 - and endothelin-1-induced besides α1 -adrenergic prostate contractions. This reflects divergent pharmacologic profiles of EHT1864 and NSC23766, although both are Rac-GTPase inhibitors. In vivo, urodynamic effects of EHT1864 and possibly of FRAX486 may exceed those of α1 -blockers.

Optimized management of urolithiasis by coloured stent-stone contrast using dual-energy computed tomography (DECT).

BACKGROUND: We analysed in vitro the appearance of commonly used ureteral stents with dual-energy computed tomography (DECT) and we used these characteristics to optimize the differentiation between stents and adjacent stone. METHODS: We analysed in vitro a selection of 36 different stents from 7 manufacturers. They were placed in a self-build phantom model and measured using the SOMATOM® Force Dual Source CT-Scanner (Siemens, Forchheim, Germany). Each sample was scanned at various tube potentials of 80 and 150 peak kilovoltage (kVp), 90 and 150 kVp and 100 and 150 kVp. The syngo Post-Processing Suite software program (Siemens, Forchheim, Germany) was used for differentiation based on a 3-material decomposition algorithm (UA, calcium, urine) according to our standard stone protocol. RESULTS: Stents composed of polyurethane appeared blue and silicon-based stents were red on the image. The determined appearances were constant for various peak kilovoltage (kVp) values. The coloured stent-stone-contrast displayed on DECT improves monitoring, especially of small calculi adjacent to indwelling ureteral stents. CONCLUSION: Both urinary calculi and ureteral stents can be accurately differentiated by a distinct appearance on DECT. For the management of urolithiasis patients can be monitored more easily and accurately using DECT if the stent shows a different colour than the adjacent stone.

Inhibition of smooth muscle contraction and ARF6 activity by the inhibitor for cytohesin GEFs, secinH3, in the human prostate.

Prostate smooth muscle contraction is critical for etiology and treatment of male lower urinary tract symptoms (LUTS) and is promoted by small monomeric GTPases (RhoA and Rac). GTPases may be activated by guanosine nucleotide exchange factors (GEFs). GEFs of the cytohesin family may indirectly activate Rac, or ADP ribosylation factor (ARF) GTPases directly. Here we investigated the expression of cytohesin family GEFs and effects of the cytohesin inhibitor Sec7 inhibitor H3 (secinH3) on smooth muscle contraction and GTPase activities in human prostate tissues. Of all four cytohesin isoforms, cytohesin-1 and -2 showed the highest expression in real-time PCR. Western blot and fluorescence staining suggested that cytohesin-2 may be the predominant isoform in prostate smooth muscle cells. Contractions induced by norepinephrine, the α1-adrenoceptor agonist phenylephrine, the thromboxane A2 analog U-46619 , and endothelin-1 and -3, as well as neurogenic contractions induced by electric field stimulation (EFS), were reduced by secinH3 (30 µM). Inhibition of EFS-induced contractions appeared to have efficacy similar to that of inhibition by the α1-adrenoceptor antagonist tamsulosin (300 nM). Combined application of secinH3 plus tamsulosin caused larger inhibition of EFS-induced contractions than tamsulosin alone. Pull-down assays demonstrated inhibition of the small monomeric GTPase ARF6 by secinH3, but no inhibition of RhoA or Rac1. In conclusion, we suggest that a cytohesin-ARF6 pathway takes part in smooth muscle contraction. This may open attractive new possibilities in medical treatment of male LUTS.

Transient receptor potential a1 (TRPA1) agonists inhibit contractions of the isolated human ureter.

AIMS: Mechanoafferent and peristaltic mechanisms of the human ureter involve transient receptor potential V1 (TRPV1)- and purinoceptor-mediated functions. Hydrogen sulphide, an endogenous TRPA1 ligand, is linked to inhibitory neurotransmission of the pig ureter. No information is available on TRPA1 activity in the human ureter. We therefore examined the distribution and function of TRPA1 in the human ureter. METHODS: Expression of TRPA1 in human ureter tissue was studied by Western blot and immunofluorescence. The TRPA1 distribution was compared to TRPV1, calcitonin gene related peptide (CGRP), tyrosine hydroxylase (TH), and vimentin. Effects of the TRPA1 agonists allyl isothiocyanate (AI), cinnamaldehyde (CA), sodium hydrogen sulfide (NaHS), and capsaicin (TRPV1 agonist) on human ureter preparations were studied in organ baths. RESULTS: By Western blot, bands were detected at the expected molecular weight for TRPA1. TRPA1- and TRPV1-immunoreactivities were located on CGRP-positive nerves, but not on TH-positive nerves. TRPA1 was also located in vimentin-positive interstitial cells. In functional experiments, neither of the TRPA1-agonists (1-100 µM) had any direct effects on ureter tension (baseline/potassium-induced contractions). However, CA, AI, NaHS, and capsaicin (10 µM) decreased (P < 0.01-0.05) tetrodotoxin-sensitive electrically induced (2,4,8,16,32 Hz) contractions. Inhibitory activities were 50-61% (CA), 30-56% (AI), 30-40% (NaHS), and 37-67% (Capsaicin). CONCLUSIONS: In the human ureter, TRPA1 is located to sensory nerves and interstitial cells. TRPA1 agonists inhibited electrically induced contractions but had no direct effect on smooth muscle tension of the human ureter. A role for TRPA1 in modulating neurotransmission and possibly peristalsis of the human ureter is proposed.

Adreno-muscarinic synergy in the male human urinary outflow tract.

AIM: To examine putative interaction between adrenergic and muscarinic contractile activation in the human urinary outflow tract. METHODS: Tissue from the trigone and prostatic urethra was obtained from 12 cystectomy and 16 prostatectomy specimen. Contractions were elicited by exposure to exogenous agonists before and after inhibition of Rho kinase and protein kinase c (PKC). Immunofluorescence and Western-blot studies were performed using antibodies to muscarinic M3-receptors (M3-R) and alpha1A-adrenoreceptors (alpha1A-AR). The study is registered with ClinicalTrials.gov, number NCT01227447. RESULTS: There was strong co-localization of M3-R and alpha1A-AR on trigonal and urethral myocytes. Western blot analysis revealed a significantly higher expression of alpha1A-AR in the superficial than in the deep trigone. Phenylephrine (PE, 1 µm) augmented contractions induced by carbachol (CA, 3 µm) to more than threefold control in the male superficial trigone, and to about sevenfold control in the proximal urethra. No such potentiation could be detected in female bladder outlet. Both PKC inhibitor GF 109203X and Rho kinase inhibitor Y-27632 reduced responses to 1 µM PE as well as to 3 µM CA significantly. However, the synergistic effect of the combined intervention remained proportionally unaffected. CONCLUSIONS: Muscarinic and adrenergic receptor activation exerts a strong synergistic effect in the male human bladder trigone and proximal urethra.

In vitro investigations of propulsion during laser lithotripsy using video tracking.

OBJECTIVES: Ureteroscopic laser lithotripsy is an important and widely used method for destroying ureter stones. It represents an alternative to ultrasonic and pneumatic lithotripsy techniques. Although these techniques have been thoroughly investigated, the influence of some physical parameters that may be relevant to further improve the treatment results is not fully understood. One crucial topic is the propulsive stone movement induced by the applied laser pulses. To simplify and speed up the optimization of laser parameters in this regard, a video tracking method was developed in connection with a vertical column setup that allows recording and subsequently analyzing the propulsive stone movement in dependence of different laser parameters in a particularly convenient and fast manner. MATERIALS AND METHODS: Pulsed laser light was applied from below to a cubic BegoStone phantom loosely guided within a vertical column setup. The video tracking method uses an algorithm to determine the vertical stone position in each frame of the recorded scene. The time-dependence of the vertical stone position is characterized by an irregular series of peaks. By analyzing the slopes of the peaks in this signal it was possible to determine the mean upward stone velocity for a whole pulse train and to compare it for different laser settings. For a proof of principle of the video tracking method, a specific pulse energy setting (1 J/pulse) was used in combination with three different pulse durations: short pulse (0.3 ms), medium pulse (0.6 ms), and long pulse (1.0 ms). The three pulse durations were compared in terms of their influence on the propulsive stone movement in terms of upward velocity. Furthermore, the propulsions induced by two different pulse energy settings (0.8 J/pulse and 1.2 J/pulse) for a fixed pulse duration (0.3 ms) were compared. A pulse repetition rate of 10 Hz was chosen for all experiments, and for each laser setting, the experiment was repeated on 15 different freshly prepared stones. The latter set of experiments was compared with the results of previous propulsion measurements performed with a pendulum setup. RESULTS: For a fixed pulse energy (1 J/pulse), the mean upward propulsion velocity increased (from 120.0 to 154.9 mm · s-1 ) with decreasing pulse duration. For fixed pulse duration (0.3 ms), the mean upward propulsion velocity increased (from 91.9 to 123.3 mm · s-1 ) with increasing pulse energy (0.8 J/pulse and 1.2 J/pulse). The latter result corresponds roughly to the one obtained with the pendulum setup (increase from 61 to 105 mm · s-1 ). While the mean propulsion velocities for the two different pulse energies were found to differ significantly (P < 0.001) for the two experimental and analysis methods, the standard deviations of the measured mean propulsion velocities were considerably smaller in case of the vertical column method with video tracking (12% and 15% for n = 15 freshly prepared stones) than in case of the pendulum method (26% and 41% for n = 50 freshly prepared stones), in spite of the considerably smaller number of experiment repetitions ("sample size") in the first case. CONCLUSION: The proposed vertical column method with video tracking appears advantageous compared to the pendulum method in terms of the statistical significance of the obtained results. This may partly be understood by the fact that the entire motion of the stones contributes to the data analysis, rather than just their maximum distance from the initial position. The key difference is, however, that the pendulum method involves only one single laser pulse in each experiment run, which renders this method rather tedious to perform. Furthermore, the video tracking method appears much better suited to model a clinical lithotripsy intervention that utilizes longer series of laser pulses at higher repetition rates. The proposed video tracking method can conveniently and quickly deliver results for a large number of laser pulses that can easily be averaged. An optimization of laser settings to achieve minimal propulsive stone movement should thus be more easily feasible with the video tracking method in connection with the vertical column setup. Lasers Surg. Med. 50:333-339, 2018. © 2017 Wiley Periodicals, Inc.

Non-Adrenergic, Tamsulosin-Insensitive Smooth Muscle Contraction is Sufficient to Replace α1 -Adrenergic Tension in the Human Prostate.

BACKGROUND: Lower urinary tract symptoms (LUTS) suggestive of benign prostatic hyperplasia may be caused by prostate smooth muscle contraction. Although α1 -blockers may improve symptoms by prostate smooth muscle relaxation, their efficacy is limited. This may be explained by non-adrenergic mediators causing contraction in parallel to α1 -adrenoceptors. However, little is known about the relevance and cooperative actions of non-adrenergic mediators in the prostate. METHODS: Prostate tissues were obtained from radical prostatectomy (n = 127 patients). Contractile responses were studied in an organ bath. RESULTS: Endothelin-1 and noradrenaline induced contractions of similar magnitude (116 ± 23 and 117 ± 18% of KCl-induced contractions). Endothelin-2- and -3-induced maximum contractions of 63 ± 8.6 and 71 ± 19% of KCl, while contractions by the thromboxane analog U46619 amounted up to 63 ± 9.4%. Dopamine-induced contractions averaged to 22 ± 4.5% of KCl, while maximum contractions by serotonin, histamine, and carbachol stayed below 10% of KCl-induced. While noradrenaline-induced contractions were inhibited by tamsulosin (300 nM), endothelin-1-, -2-, or -3-induced contraction were not. No additive effects were observed if endothelins and noradrenaline were applied consecutively to the same samples. If endothelin-1 was applied after U46619, resulting tension (172 ± 43% of KCl) significantly exceeded noradrenaline-induced contraction. Tensions following combined application of endothelin-2 or -3 with U46619 stayed below noradrenaline-induced contractions. Tension following combined application of all three endothelins with U46619 resembled maximum noradrenaline-induced tone. CONCLUSIONS: 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 α1 -adrenergic tone under therapy with α1 -blockers, explaining the limited efficacy of α1 -blockers in LUTS treatment. Contractions by thromboxane and endothelin-1 may be additive, and may exceed α1 -adrenergic tone. Prostate 77:697-707, 2017. © 2017 Wiley Periodicals, Inc.

Inhibition of Adrenergic and Non-Adrenergic Smooth Muscle Contraction in the Human Prostate by the Phosphodiesterase 10-Selective Inhibitor TC-E 5005.

BACKGROUND: The phosphodiesterase (PDE) 5 inhibitor tadalafil is available for treatment of male lower urinary tract symptoms (LUTS), while the role of other PDE isoforms for prostate smooth muscle tone is still unknown. Here, we examined effects of the PDE10-selective inhibitor TC-E 5005 on smooth muscle contraction in human prostate tissue. METHODS: Prostate samples were obtained from patients undergoing radical prostatectomy. Expression of PDE10 was addressed by RT-PCR, Western blot, and fluorescence staining with different markers. Effects of TC-E 5005 and tadalafil on contraction, and relaxation of prostate strips were studied via organ bath. RESULTS: PDE10A was detectable by RT-PCR, Western blot, and fluorescence staining in prostate tissues. Colocalization with markers suggested expression of PDE10A in smooth muscle cells and catecholaminergic nerves. Norepinephrine, the α1 -adrenergic agonist phenylephrine, the thromboxane A2 analogue U46619, and endothelins 1-3 induced concentration-dependent contractions of prostate strips, while electric field stimulation (EFS) induced frequence-dependent contractions. Application of TC-E 5005 (500 nM) caused significant inhibition of norepinephrine-, phenylephrine-, and endothelin-3-induced contractions. Inhibition of EFS-induced contractions by TC-E 5005 ranged around 50%, resembling inhibition of EFS-induced contractions by tadalafil (10 µM). The prostacyclin analog treprostinil and the nitric oxide donor DEA NONOate induced relaxations of precontracted prostate strips, which were significantly amplified by TCE 5005. CONCLUSIONS: The PDE10-selective inhibitor TC-E 5005 inhibits adrenergic and neurogenic smooth muscle contractions in the human prostate. TC-E 5005 inhibits neurogenic contractions with similar efficacy than tadalafil, so that urodynamic effects in vivo appear possible. Prostate 76:1364-1374, 2016. © 2016 Wiley Periodicals, Inc.