Non-adrenergic Contraction Research Articles

Selective inhibition of neurogenic, but not agonist-induced contractions by phospholipase A2 inhibitors points to presynaptic phospholipase A2 functions in contractile neurotransmission to human prostate smooth muscle.

Phospholipases A2 (PLA2 ) may be involved in α1 -adrenergic contraction by formation of thromboxane A2 in different smooth muscle types. However, whether this mechanism occurs with α1 -adrenergic contractions of the prostate, is still unknown. While α1 -adrenoceptor antagonists are the first line option for medical treatment of voiding symptoms in benign prostatic hyperplasia (BPH), improvements are limited, probably by nonadrenergic contractions including thromboxane A2 . Here, we examined effects of PLA2 inhibitors on contractions of human prostate tissues. Prostate tissues were obtained from radical prostatectomy. Contractions were induced by electric field stimulation (EFS) and by α1 -adrenergic agonists in an organ bath, after application of the cytosolic PLA2 inhibitors ASB14780 and AACOCF3, the secretory PLA2 inhibitor YM26734, the leukotriene receptor antagonist montelukast, or of solvent to controls. Frequency-dependent contractions of human prostate tissues induced by EFS were inhibited by 25% at 8 Hz, 38% at 16 Hz and 37% at 32 Hz by ASB14780 (1 µM), and by 32% at 16 Hz and 22% at 32 Hz by AACOCF3 (10 µM). None of both inhibitors affected contractions induced by noradrenaline, phenylephrine or methoxamine. YM26734 (3 µM) and montelukast (0.3and 1 µM) neither affected EFS-induced contractions, nor contractions by α1 -adrenergic agonists, while all contractions were substantially inhibited by silodosin (100 nM). Our findings suggest presynaptic PLA2 functions in prostate smooth muscle contraction, while contractions induced by α1 -adrenergic agonists occur PLA2 -independent. Lacking sensitivity to montelukast excludes an involvement of PLA2 -derived leukotrienes in promotion of contractile neurotransmission.

PD02-03 ARF1 GEF INHIBITOR GOLGICIDE A INHIBITS ADRENERGIC AND NON-ADRENERGIC PROSTATE SMOOTH MUSCLE CONTRACTION

You have accessJournal of UrologyCME1 Apr 2023PD02-03 ARF1 GEF INHIBITOR GOLGICIDE A INHIBITS ADRENERGIC AND NON-ADRENERGIC PROSTATE SMOOTH MUSCLE CONTRACTION Alexander Tamalunas, Amin Wendt, Ru Huang, Ruixiao Wang, Yuhan Liu, Beata Rutz, Anna Ciotkowska, Giuseppe Magistro, Christian Stief, and Martin Hennenberg Alexander TamalunasAlexander Tamalunas More articles by this author , Amin WendtAmin Wendt More articles by this author , Ru HuangRu Huang More articles by this author , Ruixiao WangRuixiao Wang More articles by this author , Yuhan LiuYuhan Liu More articles by this author , Beata RutzBeata Rutz More articles by this author , Anna CiotkowskaAnna Ciotkowska More articles by this author , Giuseppe MagistroGiuseppe Magistro More articles by this author , Christian StiefChristian Stief More articles by this author , and Martin HennenbergMartin Hennenberg More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003219.03AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Prostate smooth muscle contraction is one of the main targets for medical treatment in lower urinary tract symptoms (LUTS) secondary to benign prostatic hyperplasia (BPH). Due to an unfavorable balance between side effects and limited efficacy, current medications cause high discontinuation rates. Agonist-induced smooth muscle contractions are mediated by activation of a1-adrenoceptors, endothelin and thromboxane, and their intracellular signaling pathways including monomeric GTPases. In general, monomeric GTPases are activated by GEFs, and deactivated by GAPs. Recently, we could show, that the small monomeric GTPase ARF6 is involved in prostate smooth muscle contraction. Here, we examined the effect of the ARF1 GEF inhibitor golgicide A on agonist-induced human prostate smooth muscle contraction. METHODS: Prostate tissues were obtained from patients undergoing radical prostatectomy for prostate cancer (n=30 patients). Contractility of prostate strips was then assessed in an organ bath. Data are presented as mean ±standard deviation, with Emax and EC50 values, while group p-value is given in the diagrams. RESULTS: Noradrenaline-induced adrenergic contractions were decreased by 47±12.1% with golgicide A (10µM) for concentrations of 1-100 µM (Figure 1A), by 30±6.6% for 1-100 µM of phenylephrine-induced contractions (Figure 1B), and by 61±10.1% for 1-100 µM of methoxamine-induced contractions (Figure 1C). Additionally, golgicide A inhibited non-adrenergic contraction by thromboxane A2 analogue U46619 by 82±19.6% for 1-300 µM of U46619-induced contractions (Figure 1D), endothelin-1 induced contraction by 53±7.0% for 1-30 µM of endothelin-1-induced contractions (Figure 1E), and EFS-induced neurogenic contractions by 76±16.6% for 2-32 Hz (Figure 1F). CONCLUSIONS: Golgicide A-sensitive GEFs are involved in regulation of human prostate smooth muscle contraction, by control of ARF1 GEF activity. Knowledge about GEFs and GAPs involved in smooth muscle contraction is still limited. However, increased understanding of prostate smooth muscle contraction may assist in developing new pharmaceutical targets for LUTS medication in the future. Source of Funding: None © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e69 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Alexander Tamalunas More articles by this author Amin Wendt More articles by this author Ru Huang More articles by this author Ruixiao Wang More articles by this author Yuhan Liu More articles by this author Beata Rutz More articles by this author Anna Ciotkowska More articles by this author Giuseppe Magistro More articles by this author Christian Stief More articles by this author Martin Hennenberg More articles by this author Expand All Advertisement PDF downloadLoading ...

Inhibition of α1-Adrenergic, Non-Adrenergic and Neurogenic Human Prostate Smooth Muscle Contraction and of Stromal Cell Growth by the Isoflavones Genistein and Daidzein.

Isoflavone-rich legumes, including soy, are used for food production, as dietary supplements and in traditional medicine. Soy consumption correlates negatively with benign prostatic hyperplasia (BPH) and voiding symptoms. However, isoflavone effects on the prostate are hardly known. Here, we examined the effects on human prostate smooth muscle contractions and stromal cell growth, which are driving factors of voiding symptoms in BPH. Smooth muscle contractions were induced in prostate tissues from radical prostatectomy. Growth-related functions were studied in cultured stromal cells (WPMY-1). Neurogenic, α1-adrenergic and non-adrenergic contractions were strongly inhibited with 50 µM and by around 50% with 10 µM genistein. Daidzein inhibited neurogenic contractions using 10 and 100 µM. Agonist-induced contractions were inhibited by 100 µM but not 10 µM daidzein. A combination of 6 µM genistein with 5 µM daidzein still inhibited neurogenic and agonist-induced contractions. Proliferation of WPMY-1 cells was inhibited by genistein (>50%) and daidzein (<50%). Genistein induced apoptosis and cell death (by seven-fold relative to controls), while daidzein induced cell death (6.4-fold) without apoptosis. Viability was reduced by genistein (maximum: 87%) and daidzein (62%). In conclusion, soy isoflavones exert sustained effects on prostate smooth muscle contractions and stromal cell growth, which may explain the inverse relationships between soy-rich nutrition, BPH and voiding symptoms.

A0599 - WNK463 and HTH01-015 simultaneously inhibit neurogenic and α1-blocker-resistant, non-adrenergic contractions of human prostate tissues, and growth-related functions of prostate stromal cells

A0599 - WNK463 and HTH01-015 simultaneously inhibit neurogenic and α1-blocker-resistant, non-adrenergic contractions of human prostate tissues, and growth-related functions of prostate stromal cells

PD11-07 MIRABEGRON INHIBITS NEUROGENIC AND α 1 -ADRENERGIC HUMAN PROSTATE SMOOTH MUSCLE CONTRACTIONS, WITH SIMILAR PHARMACOLOGIC PATTERNS AS α 1 -BLOCKERS

You have accessJournal of UrologyBenign Prostatic Hyperplasia: Basic Research & Pathophysiology (PD11)1 Sep 2021PD11-07 MIRABEGRON INHIBITS NEUROGENIC AND α1-ADRENERGIC HUMAN PROSTATE SMOOTH MUSCLE CONTRACTIONS, WITH SIMILAR PHARMACOLOGIC PATTERNS AS α1-BLOCKERS Ru Huang, Yuhan Liu, Bingsheng Li, Ruixiao Wang, Alexander Tamalunas, Raphaela Waidelich, Frank Strittmatter, Christian G. Stief, and Martin Hennenberg Ru HuangRu Huang More articles by this author , Yuhan LiuYuhan Liu More articles by this author , Bingsheng LiBingsheng Li More articles by this author , Ruixiao WangRuixiao Wang More articles by this author , Alexander TamalunasAlexander Tamalunas More articles by this author , Raphaela WaidelichRaphaela Waidelich More articles by this author , Frank StrittmatterFrank Strittmatter More articles by this author , Christian G. StiefChristian G. Stief More articles by this author , and Martin HennenbergMartin Hennenberg More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000001986.07AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Mirabegron inhibits detrusor smooth muscle contractions and is available for treatment of overactive bladder. Consequently, its effects on lower urinary tract symptoms (LUTS) suggestive of bengin prostatic hyperplasia (BPH) were subject of recent trials. However, available results are not yet conclusive, and effects of mirabegron on human prostate smooth muscle contraction are hardly characterized. Voiding symptoms suggestive of BPH may be driven by increased prostate smooth muscle tone. Here, we examined effects of mirabegron on contractions of human prostate tissues. METHODS: Human prostate tissues were obtained from radical prostatectomy. Contractions were studied in an organ bath. Comparisons were performed between whole groups (one-way ANOVA), and at single frequencies and agonist concentrations (multivariate analysis). RESULTS: Electric field stimulation (EFS) induced neurogenic, frequence-dependent contractions, while agonists induced concentration-dependent contractions. Mirabegron (10 µM) inhibited EFS-induced contractions (p <0.001 for controls vs. mirabegron between whole groups), which was observed at different frequencies and reflected by reduced Emax values calculated by curve fitting (contractions in controls 84±11% of KCl-induced contractions, but 14±2% after mirabegron, p <0.01). Miragebron increased the EC50 values for noradrenaline (controls 0.55±0.32 µM, mirabegron 5.1±2.4 µM, p <0.004), resulting in rightshifts of concentration response curves for noradrenaline-induced contractions (p <0.001) and in reduced contractions up to 10 µM noradrenaline, but in recovery at higher noradrenaline concentrations and unchanged Emax values. Similarly, EC50 values for the α1-adrenergic agonist phenylephrine were increased by mirabegron (controls 7.4±4.1 µM, mirabegron 29.2±8.9 µM, p=0.05), resulting in rightshifts of concentration response curves for phenylephrine (p <0.001) and in reduced contractions up to 30 µM phenylephrine, but in recovery at higher phenylephrine concentrations and in unchanged Emax values. Mirabegron slightly reduced contractions induced by the thromboxane A2 analog U46619 (p <0.03), without changing EC50 values. Contractions induced by endothelin-1 remained unchanged by mirabegron. CONCLUSIONS: Mirabegron inhibits neurogenic and α1-adrenergic smooth muscle contractions in the human prostate. Surprisingly, the pharmacologic pattern is similar to that of α1-blockers, including rightshifts of α1-adrenergic concentration response curves, reduced Emax values for neurogenic contractions, and lacking or small effects on non-adrenergic contractions. Source of Funding: China Scholarship Council (CSC) © 2021 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 206Issue Supplement 3September 2021Page: e200-e200 Advertisement Copyright & Permissions© 2021 by American Urological Association Education and Research, Inc.MetricsAuthor Information Ru Huang More articles by this author Yuhan Liu More articles by this author Bingsheng Li More articles by this author Ruixiao Wang More articles by this author Alexander Tamalunas More articles by this author Raphaela Waidelich More articles by this author Frank Strittmatter More articles by this author Christian G. Stief More articles by this author Martin Hennenberg More articles by this author Expand All Advertisement Loading ...

Lenalidomide and pomalidomide inhibit growth of prostate stromal cells and human prostate smooth muscle contraction

AimsMedical treatment for lower urinary tract symptoms secondary to benign prostatic hyperplasia is characterized by an unfavorable balance between limited efficacy and pronounced side effects. We recently reported, that thalidomide reduces prostate smooth muscle contraction and inhibits cell growth. Like thalidomide, its analogs lenalidomide and pomalidomide are also in clinical use. Therefore, we investigated the effects of lenalidomide and pomalidomide on human prostate smooth muscle contraction, cytoskeletal organization, and growth-related functions in stromal cells. Materials and methodsProliferation was assessed by EdU assay and colony formation, cytoskeletal organization by phalloidin staining, cell viability by CCK8, and apoptosis and cell death by flow cytometry in cultured prostate stromal cells (WPMY-1). Contractions of human prostate tissues from radical prostatectomy were induced by methoxamine, noradrenaline, phenylephrine, endothelin-1, U46619, or electric field stimulation (EFS) in an organ bath. Key findingsProliferation of WPMY-1 cells was significantly reduced by lenalidomide (5–200 μM) and pomalidomide (2.5–5 μM). In parallel, organization of actin filaments collapsed after treatment with lenalidomide and pomalidomide. Lenalidomide and pomalidomide inhibited both adrenergic contractions and non-adrenergic contractions as well as neurogenic contractions induced by EFS. Neither reduction in viability, nor increase in cell death or apoptosis was observed in WPMY-1 cells. SignificanceThalidomide-derivatives impair growth of human prostate stromal cells, without showing a decrease in cell viability and, in parallel, inhibit adrenergic, neurogenic, and non-adrenergic contractions by breakdown of the actin cytoskeleton. Urodynamic effects in vivo appear possible.

Concentration-dependent alpha1-Adrenoceptor Antagonism and Inhibition of Neurogenic Smooth Muscle Contraction by Mirabegron in the Human Prostate

Introduction: Mirabegron is available for treatment of storage symptoms in overactive bladder, which may be improved by β3-adrenoceptor-induced bladder smooth muscle relaxation. In addition to storage symptoms, lower urinary tract symptoms in men include obstructive symptoms attributed to benign prostatic hyperplasia, caused by increased prostate smooth muscle tone and prostate enlargement. In contrast to the bladder and storage symptoms, effects of mirabegron on prostate smooth muscle contraction and obstructive symptoms are poorly understood. Evidence from non-human smooth muscle suggested antagonism of α1-adrenoceptors as an important off-target effect of mirabegron. As α1-adrenergic contraction is crucial in pathophysiology and medical treatment of obstructive symptoms, we here examined effects of mirabegron on contractions of human prostate tissues and on proliferation of prostate stromal cells. Methods: Contractions were induced in an organ bath. Effects of mirabegron on proliferation, viability, and cAMP levels in cultured stromal cells were examined by EdU assays, CCK-8 assays and enzyme-linked immunosorbent assay. Results: Mirabegron in concentrations of 5 and 10 μM, but not 1 µM inhibited electric field stimulation-induced contractions of human prostate tissues. Mirabegron in concentrations of 5 and 10 µM shifted concentration response curves for noradrenaline-, methoxamine- and phenylephrine-induced contractions to the right, including recovery of contractions at high concentrations of α1-adrenergic agonists, increased EC50 values, but unchanged Emax values. Rightshifts of noradrenaline concentration response curves and inhibition of EFS-induced contractions were resistant to L-748,337, l-NAME, and BPIPP. 1 µM mirabegron was without effect on α1-adrenergic contractions. Endothelin-1- and U46619-induced contractions were not affected or only inhibited to neglectable extent. Effects of mirabegron (0.5–10 µM) on proliferation and viability of stromal cells were neglectable or small, reaching maximum decreases of 8% in proliferation assays and 17% in viability assays. Mirabegron did not induce detectable increases of cAMP levels in cultured stromal cells. Conclusion: Mirabegron inhibits neurogenic and α1-adrenergic human prostate smooth muscle contractions. This inhibition may be based on antagonism of α1-adrenoceptors by mirabegron, and does not include activation of β3-adrenoceptors and requires concentrations ranging 50-100fold higher than plasma concentrations reported from normal dosing. Non-adrenergic contractions and proliferation of prostate stromal cells are not inhibited by mirabegron.

Inhibition of human prostate stromal cell growth and smooth muscle contraction by thalidomide: A novel remedy in LUTS?

Medical treatment in benign prostatic hyperplasiatargets prostate size to prevent disease progression, complications, and surgery, and prostate smooth muscle tone for rapid relief of lower urinary tract symptoms. Combination therapies are still required to target both at once. However, current medications are insufficient, due to an unfavorable balance between side effects and efficacy. The limited efficacy of α1 -blockers may be due to nonadrenergic mediators like endothelin-1 and thromboxane A2 (TXA2 ), which keep up prostate smooth muscle contraction even in the presence of α1 -blockers. Consequently, future options with higher efficacy need to target α1 -adrenergic and nonadrenergic contractions as well as stromal cell growth at once. Thalidomide has been approved as an oral medication for various diseases, including the treatment of prostate cancer. Therefore, we investigated the effect of thalidomide on cellular functions of prostate stromal cells and human prostate smooth muscle contraction. Cytoskeletal organization was visualized by phalloidin staining, cell growth was assessed by 5-ethynyl-2'-deoxyuridine assay, cell viability by cell counting kit-8, and apoptosis and cell death by flow cytometry in cultured prostate stromal cells (WPMY-1). Contractions of human prostate tissues from radical prostatectomy were studied in an organ bath, where they were induced by the α1 -adrenoceptor agonists methoxamine, noradrenaline, phenylephrine, and the nonadrenergic agonists endothelin-1, TXA2 analog U46619, or electric field stimulation (EFS). Thalidomide significantly reduced the proliferation of WPMY-1 cells, which was time- and concentration-dependent (10-300 µM). In parallel, organization of actin filaments collapsed after treatment with thalidomide. Thalidomide (30-100 µM) inhibited noradrenaline-, phenylephrine-, and methoxamine-induced contractions, as well as nonadrenergic contractions induced by endothelin-1 and U46619, and neurogenic contractions induced by EFS. No reduction in viability and no increases in apoptosis or in cell death were observed in WPMY-1 cells. Thalidomide impairs the growth of human prostate stromal cells, without showing a decrease in cell viability. In parallel, thalidomide inhibits adrenergic, neurogenic, and nonadrenergic contractions. This may be explained by a breakdown of the actin cytoskeleton. In vivo, urodynamic effects of thalidomide appear possible and may even exceed those of α1 -blockers or combination therapies.

MP06-14 INHIBITION OF PROSTATE SMOOTH MUSCLE CONTRACTION BY THALIDOMIDE: A NOVEL REMEDY IN LUTS?

You have accessJournal of UrologyBenign Prostatic Hyperplasia: Basic Research & Pathophysiology (MP06)1 Apr 2020MP06-14 INHIBITION OF PROSTATE SMOOTH MUSCLE CONTRACTION BY THALIDOMIDE: A NOVEL REMEDY IN LUTS? Alexander Tamalunas*, Cora Sauckel, Anna Ciotkowska, Christian Gratzke, Christian G. Stief, and Martin Hennenberg Alexander Tamalunas*Alexander Tamalunas* More articles by this author , Cora SauckelCora Sauckel More articles by this author , Anna CiotkowskaAnna Ciotkowska More articles by this author , Christian GratzkeChristian Gratzke More articles by this author , Christian G. StiefChristian G. Stief More articles by this author , and Martin HennenbergMartin Hennenberg More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000000820.014AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Medical treatment in benign prostatic hyperplasia (BPH) includes reduction of smooth muscle tone for rapid relieve of lower urinary tract symptoms (LUTS). Current medications cause high rates of patient non-compliance due to an unfavourable balance between side effects and efficacy, with discontinuation rates peaking up to 90% for combination therapies. We have recently shown that thalidomide reduces prostate stromal cell growth and modulates cytoskeletal actin organization. Here, we addressed effects of thalidomide on adrenergic and non-adrenergic contractions of human prostate smooth muscle. METHODS: Prostate tissues were obtained from patients undergoing radical prostatectomy for prostate cancer (n=40 patients). Contractility of prostate strips was then assessed in an organ bath. RESULTS: Noradrenaline-induced adrenergic contractions were decreased up to over 56 % with thalidomide (100µM) for concentrations of 1-100 µM (p<0.05, p<0.03, p<0.02, p<0.01, p<0.02 for 1 µM, 3 µM, 10 µM, 30 µM and 100 µM noradrenaline, respectively, for thalidomide vs. control, fig. A). Thalidomide caused significant inhibition of up to 90 % at 1-100 µM for both methoxamine- and phenylephrine-induced contractions (p<0.04, p<0.03, p<0.04, p<0.04, p<0.03 and p<0.05, p<0.02, p<0.03, p<0.02, p<0.01 for 1 µM, 3 µM, 10 µM, 30 µM and 100 µM phenylephrine and methoxamine, respectively, for thalidomide vs. control, fig. B & C). Thalidomide inhibited non-adrenergic contraction with thromboxane A2 analogue U46619 at 0.1 µM, 0.3 µM, 3 µM, 10 µM and 30 µM (p<0.01 for 0.1 µM, 0.3 µM, 10 µM and 30 µM and p<0.02 for 3 µM U46619, respectively, for thalidomide vs. control) and up to 58 % of endothelin-1-induced contractions at 0.3 µM, 1 µM and 3 µM (p<0.02, p<0.03 and p<0.03 for 0.3 µM, 1 µM and 3 µM endothelin-1, respectively, for thalidomide vs. control, fig. D & E). Significant inhibition up to 73 % also occurred in EFS-induced neurogenic contractions for 2-32 Hz (p<0.01, p<0.05, p<0.04, p<0.03, p<0.03 for 2, 4, 8, 16 and 32 Hz, respectively, for thalidomide vs. control, fig. F). CONCLUSIONS: Thalidomide not only inhibits prostate stromal cell growth and modulates cytoskeletal development, but also inhibits adrenergic and non-adrenergic prostate smooth muscle contraction. Therefore, thalidomide may be superior to current medical treatment. Source of Funding: None. © 2020 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 203Issue Supplement 4April 2020Page: e56-e56 Advertisement Copyright & Permissions© 2020 by American Urological Association Education and Research, Inc.MetricsAuthor Information Alexander Tamalunas* More articles by this author Cora Sauckel More articles by this author Anna Ciotkowska More articles by this author Christian Gratzke More articles by this author Christian G. Stief More articles by this author Martin Hennenberg More articles by this author Expand All Advertisement PDF downloadLoading ...

The STK16 inhibitor STK16-IN-1 inhibits non-adrenergic and non-neurogenic smooth muscle contractions in the human prostate and the human male detrusor.

Mixed lower urinary tract symptoms (LUTS) (voiding symptoms suggestive of benign prostatic hyperplasia plus storage symptoms, which can be caused by overactive bladder) are common in men. Unwanted contraction of prostate and/or bladder smooth muscle has been implied in the pathophysiology of male LUTS. Here, we examined effects of the serine/threonine kinase 16 (STK16) inhibitor STK16-IN-1 on contraction of human tissues from the prostate and male detrusor. Tissues were obtained from radical prostatectomy and radical cystectomy. Contractions were studied in an organ bath and STK16 expressions by Western blot analyses and fluorescence staining. In prostate tissues, STK16-IN-1 (1μM) inhibited contractions induced by endothelin-1 and the thromboxane A2 analog U46619. Contractions of prostate tissues induced by noradrenaline, the α1-agonists phenylephrine and methoxamine, or electric field stimulation (EFS) were not changed by STK16-IN-1. In male detrusor tissues, STK16-IN-1 inhibited contractions induced by the cholinergic agonists carbachol and metacholine, and contractions induced by U46619. EFS-induced contractions of detrusor tissues were not changed by STK16-IN-1. Western blot analyses of prostate and detrusor tissues revealed bands matching the molecular weight of STK16. Fluorescence staining of prostate tissues using STK16 antibodies resulted in immunoreactivity in smooth muscle cells.STK16-IN-1 selectively inhibits non-adrenergic/non-neurogenic smooth muscle contractions in the male prostate and to limited extent in the bladder. Because non-adrenergic contractions in the male LUTS may account for limited efficacy of α1-blockers and for α1-blocker-resistant symptoms, studies assessing add-on of STK16-IN-1 to α1-blockers in mixed LUTS appear feasible.

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

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. 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. 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. 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.

MP45-11 NEW STRATEGIES FOR INHIBITION OF NON-ADRENERGIC PROSTATE SMOOTH MUSCLE CONTRACTION BY PHARMACOLOGIC INTERVENTION

You have accessJournal of UrologyBenign Prostatic Hyperplasia: Basic Research & Pathophysiology1 Apr 2018MP45-11 NEW STRATEGIES FOR INHIBITION OF NON-ADRENERGIC PROSTATE SMOOTH MUSCLE CONTRACTION BY PHARMACOLOGIC INTERVENTION Qingfeng Yu, Christian Gratzke, Paul Kuppermann, Annika Herlemann, Yiming Wang, Frank Strittmatter, Raphaela Waidelich, Christian Stief, and Martin Hennenberg Qingfeng YuQingfeng Yu More articles by this author , Christian GratzkeChristian Gratzke More articles by this author , Paul KuppermannPaul Kuppermann More articles by this author , Annika HerlemannAnnika Herlemann More articles by this author , Yiming WangYiming Wang More articles by this author , Frank StrittmatterFrank Strittmatter More articles by this author , Raphaela WaidelichRaphaela Waidelich More articles by this author , Christian StiefChristian Stief More articles by this author , and Martin HennenbergMartin Hennenberg More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2018.02.1450AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Inhibition of prostate smooth muscle contraction by a1-adrenoceptor antagonists (a1-blockers) is the first option for medical treatment of lower urinary tract symptoms (LUTS) suggestive of benign prostatic hyperplasia (BPH). In fact, increased prostate smooth muscle tone may drive urethral obstruction and LUTS. However, the efficacy of α1-blockers is limited to 50%, because non-adrenergic mediators including endothelin-1 and thromboxane A2 (TXA2) increase prostate smooth muscle tension in parallel to α1-adrenoceptors and may therefore keep urethral obstruction despite therapy with α1-blockers. Consequently, future options with highest efficacy need to target α1-adrenergic and non-adrenergic contractions at once. Recently, several compounds have been reported to inhibit adrenergic prostate contraction, but their effects on non-adrenergic contraction are still unknown. Here, we examined effects of Rac-GTPase inhibitors and of a Src family kinase (SFK) inhibitor on non-adrenergic prostate contractions. METHODS Prostate tissues were obtained from radical prostatectomy. Contractions were studied in an organ bath, where they were induced by the α1-adrenoceptor agonist methoxamine, the TXA2 analog U46619, or by endothelin-1. RESULTS Methoxamine, U46619, and endothelin-1 induced concentration-dependent contractions of prostate strips. Inhibitory effects of the Rac inhibitors EHT1864 (100 µM) and NSC23766 (100 µM) and of the SFK inhibitor AZM475721 (10 µM) on α1-adrenergic contraction were confirmed by inhibition of methoxamine-induced contractions by all three inhibitors. EHT1864 caused significant inhibition of endothelin-1- and U46619-induced contractions, which was observed at 0.1-3 μM of endothelin-1 (p<0.001 between EHT1864 and control group), and at 1-30 μM of U46619 (p<0.001 between EHT1864 and control group). NSC23766 caused significant inhibition of U46619-induced contractions, which was observed at 3-10 µM of U46619 (p<0.001 between NSC23766 and control group). NSC23766 did not change endothelin-1-induced contractions. AZM475721 did not change U46619- or endothelin-1-induced contractions. CONCLUSIONS From the three examined inhibitors, EHT1864 is the most promising from a translational 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, but not of AZM475721 may exceed those of α1-blockers. © 2018FiguresReferencesRelatedDetails Volume 199Issue 4SApril 2018Page: e601-e602 Advertisement Copyright & Permissions© 2018MetricsAuthor Information Qingfeng Yu More articles by this author Christian Gratzke More articles by this author Paul Kuppermann More articles by this author Annika Herlemann More articles by this author Yiming Wang More articles by this author Frank Strittmatter More articles by this author Raphaela Waidelich More articles by this author Christian Stief More articles by this author Martin Hennenberg More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Evidence for sympathetic, purinergic transmission in the iris dilator muscle of the rabbit.

Electrical transmural stimulation of isolated iris dilator muscle of the rabbit produced a transient contraction that consisted of adrenergic and nonadrenergic components. In contrast to the adrenergic component, the nonadrenergic component was resistant to prazosin and other adrenoceptor antagonists. However, both components were completely blocked by guanethidine or tetrodotoxin. Among some tested compounds including neuropeptide Y, both ATP and 2-methylthio ATP produced a transient contraction in the dilator muscle and the sustained treatment with each markedly attenuated the nonadrenergic responses to electrical stimulation and to ATP. Suramin had no effect on and alpha,beta-methylene ATP potentiated the responses to electrical stimulation and to ATP. These results suggest that the nonadrenergic contraction induced by electrical transmural stimulation is a sympathetic purinergic response that may be mediated through unique purinoceptors.

Indirect evidence for separate vesicular neuronal origins of norepinephrine and ATP in the rabbit vas deferens

Various modulators of neurotransmission were examined for selective effects on the non-adrenergic or adrenergic components of neurotransmission in the vas deferens to test the hypothesis that ATP and norepinephrine are secreted from the same vesicles. The ATP receptor antagonist, arylazido aminopropionyl ATP (ANAPP), selectively depressed the non-adrenergic contraction and prazosin selectivity depressed the adrenergic contraction in response to electrical stimulation. These results are consistent with the presence of two neurotransmitters, ATP and norepinephrine, which mediate neurogenic contractions. Prostaglandin E 2 inhibited non-adrenergic, but enhanced adrenergic, electrically induced (10 Hz) contractions, presumably via a prejunctional mechanism. The adrenergic component of the neurogenic response was significantly more sensitive to treatment with guanethidine, guanabenz, and 6-hydroxydopamine. These results with a variety of agents are inconsistent with the hypothesis that the neurotransmitters, ATP and norepineprine, are released in tandem from the same neuronal granules.

Contribution of galanin to non‐cholinergic, non‐adrenergic transmission in rat ileum

1. We examined the possibility that the neuropeptide, galanin, may act as a transmitter in longitudinal muscle isolated from the rat ileum. 2. Galanin at nanomolar concentrations produced a phasic contraction with a concomitant increase in rhythmic activity. At concentrations in excess of 3 x 10(-8) M, the contraction was followed by a rapid desensitization; hence, with the cumulative re-addition of galanin, there was no response. This desensitization was probably selective for galanin because there was no attenuation of the contractile responses to substance P, neurokinin A and B, bradykinin or carbachol. 3. The phasic contraction induced by galanin was not inhibited by atropine, guanethidine, hexamethonium, naloxone, tetrodotoxin or [D-Pro2, D-Trp7,9]-substance P. 4. Electrical stimulation of intramural nerves at low frequencies (1-5 Hz) led to an augmentation of spontaneous rhythmic contractions, which were completely or partially inhibited by atropine. However, guanethidine, hexamethonium, naloxone, [D-Pro2, D-Trp7,9]-substance P and desensitization to galanin were without effect on the response to such electrical stimulation. 5. In contrast, transmural electrical stimulation at higher frequencies in the presence of atropine and guanethidine produced biphasic contractile responses with transient and slow components. The slow component was selectively attenuated by galanin desensitization. 6. The slow component induced by high frequency stimulation was markedly attenuated by repeated electrical stimulation at short intervals (2.5 min between 30 s trains). Following repeated stimulation, the contractile response to galanin was also attenuated. Thus, a cross-desensitization between the mediator of the slow component and galanin had to be considered. In contrast, responses to tachykinins and the transient component induced by electrical stimulation were without effect. 7. Somatostatin, vasoactive intestinal polypeptide and alpha,beta-methylene ATP were without effect on the tone of the muscle. Calcitonin gene-related peptide, neurotensin, gastrin-releasing peptide, neuropeptide Y and capsaicin produced either a transient arrest of the spontaneous rhythmic activity or a transient relaxation. 8. These results suggest that the slow component of the non-cholinergic non-adrenergic contraction, as induced by intramural nerve stimulation is apparently due to the endogenous release of galanin, presumably released from galanin-containing nerves in the rat ileum.

Pharmacological characteristics of the postsynaptically mediated contractile responses of guinea-pig ileum to long-lasting electrical field stimulation

The contractile responses of the longitudinal smooth muscle layer of the isolated guinea-pig ileum which were elicited by long-lasting (40 sec train duration) electrical field stimulation (0.8 msec, 20 V), applied at a frequency of either 5 or 30 Hz, comprised an initial phasic component followed by a secondary tonic contraction, were studied against the background of postsynaptically-acting receptor antagonists. Atropine and apamin reduced both components of the responses while prazosin produced no change. Propranolol induced a slight reduction, decreasing mainly the responses evoked by stimulation at a frequency of 5 Hz. The simultaneous application of cholinoceptor and adrenoceptor Mockers revealed a non-cholinergic, non-adrenergic component of the responses. The putative prostaglandin antagonist, SC 19220 was found to reduce mainly the tonic component of the non-cholinergic, non-adrenergic contractions evoked by stimulation at both frequencies used. These results demonstrated that the two-component contractile responses of the guinea pig ileum to long-lasting electrical stimulation were due to the release of more than one neurotransmitter and/or spasmogenic substance. It is concluded that the initial phasic component was evoked by acetylcholine and by a non-cholinergic, non-adrenergic neurotransmitter, while the tonic component was maintained predominantly by prostaglandins released during stimulation.

O-67 - Galanin mediates noncholinergic, nonadrenergic contraction induced by intramural nerve stimulation in rat ileum

O-67 - Galanin mediates noncholinergic, nonadrenergic contraction induced by intramural nerve stimulation in rat ileum

Evidence for sympathetic, purinergic transmission in the mesenteric artery of the dog.

Electrical transmural stimulation of isolated mesenteric artery of the dog produced a transient contraction which consisted of adrenergic and non-adrenergic components. In contrast to the adrenergic component, the nonadrenergic component was resistant to prazosin and other adrenoceptor-blocking agents. However, the nonadrenergic component was completely blocked by guanethidine and by desensitization with alpha,beteta-methylene-ATP (alpha,beta-MeATP). Desensitization induced by alpha,beta-MeATP also inhibited the contractile response to ATP but not the adrenergic responses induced by electrical transmural stimulation and exogenous noradrenaline. These results suggest that the nonadrenergic contraction induced by electrical transmural stimulation is a sympathetic, purinergic response.

Adrenergic and Non-Adrenergic Contraction of Isolated Urethral Muscle from Rabbit and Man

Isolated urethral muscle from rabbit and man was subjected to electrical field stimulation and the components of the resulting contraction were analyzed. Contraction usually consisted of 2 main components. One was rapidly developing, non-adrenergic and atropine-resistant. The other developed more slowly and was sensitive to α-adrenoceptor blockade and to chemical sympathectomy with 6-hydroxydopamine. By lowering the temperature from 37C to 20C, these contraction components could be separated from each other. Both could be blocked by tetrodotoxin but the effects of this drug were not consistent in human tissue. Characteristic for the initial contraction component was its dependence on the tension of the preparation; it increased with increasing tension. The adrenergic part of the contraction could be effectively blocked by phentolamine and prazosin, whereas rauwolscine was less effective. Atropine and scopolamine also inhibited the adrenergic part of the contraction, whereas acetylcholine caused a transient increase. The non-adrenergic contraction component was less sensitive to deprivation of extracellular Ca2+ than the adrenergic; almost 40 per cent remained after exposure to Ca2+ free solution for 40min., whereas the adrenergic component disappeared after 20 min. exposure. Light and electron microscopic investigation revealed 3 distinct layers of the rabbit urethral wall, 1 outer consisting of smooth and striated muscle, 1 middle consisting of smooth muscle only, and a submucosal layer where vessels and smooth muscle cells were found. Removal of the longitudinal muscle layer did not change the responses to electrical stimulation. The results suggest that the electrically induced adrenergic activation of urethral muscle of both rabbit and man was mediated mainly via α-adrenoceptors and that this muscle is innervated not only by sympathetic, adrenergic nerves but also by a type of nerve able to release a non-cholinergic, contraction-mediating transmitter.

Possible involvement of adenine nucleotides in the neurotransmission of the mouse urinary bladder

1. The urinary bladder of the mouse contracts to several agonists, namely acetylcholine, noradrenaline, adrenaline, histamine, angiotensin, serotonin, purine nucleotides and prostaglandin F 2α. 2. Atropine partially reduced the contraction induced by electrical stimulation, whereas propranolol and tolazoline were ineffective. 3. The atropine resistant component of the neurogenic response was reduced by indomethacin. Methysergide and diphenhydramine were ineffective. 4. Desensitization of the bladder by α,β-methylene ATP abolished the response to ATP and greatly reduced the non-adrenergic non-cholinergic component of the neurogenic response. 5. The results suggest that ATP could be the transmitter responsible for the non-cholinergic non-adrenergic contraction of the mouse urinary bladder.