EFS-induced Contractions Research Articles

Molecular Expression and Pharmacological Evidence for a Functional Role of Kv7 Channel Subtypes in Guinea Pig Urinary Bladder Smooth Muscle

Voltage-gated Kv7 (KCNQ) channels are emerging as essential regulators of smooth muscle excitability and contractility. However, their physiological role in detrusor smooth muscle (DSM) remains to be elucidated. Here, we explored the molecular expression and function of Kv7 channel subtypes in guinea pig DSM by RT-PCR, qRT-PCR, immunohistochemistry, electrophysiology, and isometric tension recordings. In whole DSM tissue, mRNAs for all Kv7 channel subtypes were detected in a rank order: Kv7.1~Kv7.2Kv7.3~Kv7.5Kv7.4. In contrast, freshly-isolated DSM cells showed mRNA expression of: Kv7.1~Kv7.2Kv7.5Kv7.3~Kv7.4. Immunohistochemical confocal microscopy analyses of DSM, conducted by using co-labeling of Kv7 channel subtype-specific antibodies and α-smooth muscle actin, detected protein expression for all Kv7 channel subtypes, except for the Kv7.4, in DSM cells. L-364373 (R-L3), a Kv7.1 channel activator, and retigabine, a Kv7.2-7.5 channel activator, inhibited spontaneous phasic contractions and the 10-Hz electrical field stimulation (EFS)-induced contractions of DSM isolated strips. Linopiridine and XE991, two pan-Kv7 (effective at Kv7.1-Kv7.5 subtypes) channel inhibitors, had opposite effects increasing DSM spontaneous phasic and 10 Hz EFS-induced contractions. EFS-induced DSM contractions generated by a wide range of stimulation frequencies were decreased by L-364373 (10 µM) or retigabine (10 µM), and increased by XE991 (10 µM). Retigabine (10 µM) induced hyperpolarization and inhibited spontaneous action potentials in freshly-isolated DSM cells. In summary, Kv7 channel subtypes are expressed at mRNA and protein levels in guinea pig DSM cells. Their pharmacological modulation can control DSM contractility and excitability; therefore, Kv7 channel subtypes provide potential novel therapeutic targets for urinary bladder dysfunction.

The receptor antagonist picotamide inhibits adrenergic and thromboxane-induced contraction of hyperplastic human prostate smooth muscle

Inhibition of prostate smooth muscle contraction is an important strategy for medical treatment of lower urinary tract symptoms (LUTS). Besides α1-adrenoceptors, prostate smooth muscle contraction is induced by activation of thromboxane (TXA2) receptors (TXA2-R). Here, we examined the effects of the TXA2-R antagonist picotamide on contraction of human prostate tissue. Prostate tissues were obtained from radical prostatectomy. The effects of picotamide (300 μM), L-665,240 (3 μM), and seratrodast (3 μM) on U46619-, electric field stimulation- (EFS-), phenylephrine-, and norepinephrine-induced contractions were studied in organ baths. Expression of TXA2-R and TXA2 synthase (TXS) was examined by fluorescence stainings. Picotamide, seratrodast, and L-655,240 inhibited concentration-dependent contractions induced by the TXA2 analog U46619. Picotamide, but not seratrodast or L-655,240, inhibited frequency-dependent contractions induced by EFS. Picotamide inhibited concentration-dependent contractions induced by norepinephrine or by the selective α1-adrenoceptor agonist phenylephrine. In prostate strips, where only submaximal contraction by a low dose of phenylephrine was induced, application of U46619 raised tone to maximum phenylephrine-induced tension. Immunoreactivity for TXA2-R and TXS was observed in the stroma and in epithelial cells of glands. Colocalization of both immunoreactivites was observed with the smooth muscle markers calponin and α-smooth muscle actin, with the epithelial marker pan-cytokeratin, and with prostate-specific antigen in the stroma and glands. The receptor antagonist picotamide inhibits α1-adrenergic, TXA2-mediated, and EFS-induced contractions in the human prostate. To the best of our knowledge, this is the first antagonist able to inhibit two different contraction systems in the prostate.

Functional bombesin receptors in urinary tract of rats and human but not of pigs and mice, an in vitro study

AimsBombesin receptors (BB receptors) and/or bombesin related peptides are expressed in the lower urinary tract, though their function and distribution in different species is largely unknown. This study examines whether BB receptor agonists can contract bladder smooth muscle in rats, mice, pigs and humans. MethodsBladder strips were placed in tissue baths for in vitro contractility. Neuronally evoked contractions were elicited using electric field stimulation (EFS). Effects of the BB receptor agonists, neuromedin B (NMB; BB1 receptor agonist) and gastrin-releasing peptide (GRP; BB2 receptor agonist) on baseline tone and EFS-induced contractions were monitored. ResultsIn rat and human bladder strips, NMB and GRP (10−11–10−6M) increased EFS-induced contractions in a concentration dependent manner. In these species, NMB and GRP also increased baseline tension. In mouse and pig bladder strips, NMB and GRP (10−8–3×10−6M) had no effects on either parameter. ConclusionsThese data suggest that bombesin receptors BB receptor 1 and/or BB receptor 2 increase bladder contractions in rat and human. The site of action of these receptors may be pre- and/or post-synaptic, increasing release of transmitters or enhancing smooth muscle excitability, respectively. Thus, BB1 receptor and/or BB2 receptor may offer therapeutic targets for voiding dysfunction associated with impaired bladder contractility; however, species differences must be considered when studying these receptors.Part of this work was published in an abstract form at the SFN meeting New Orleans, 2012.

Frequency dependence of excitation–contraction of multicellular smooth muscle preparations: the relevance to bipolar electrosurgery

BackgroundBipolar electrosurgical tissue welding uses forceps-like electrodes for grasping the tissues and delivering high-frequency electric current (HFEC) to produce local heat, desiccation, and protein denaturation, resulting in the fusion of the contacting tissues. Although in this technique no electric current is flowing through the whole body to cause electric injury, depending on the frequency of applied energy, it may produce local excitation of intramural nerves, which can propagate beyond the surgical site potentially causing harmful effects. Materials and methodsThe effects of varying frequency of HFEC on tissue excitability in bipolar electrosurgical modality were studied in vitro using electric field stimulation (EFS) method on multicellular smooth muscle strips of rat vas deferens. Contractile response to 5-s-long sine wave EFS train was taken as the measure of excitation of intramural nerves. ResultsEFS-induced contraction consisted of phasic and tonic components. The amplitude of both components decreased with increasing frequency, with tonic component disappearing at about 10 kHz and phasic component at about 50 kHz. Because components of EFS-induced contraction depend on different neurotransmitters, this indicates that various neurotransmitter systems are characterized by distinct frequency dependence, but above 50 kHz they all become inactivated. Bipolar electrosurgical sealing of porcine gut showed no difference in the structure of seal area at HFEC of 67 and 533 kHz. ConclusionsEFS frequency of 50 kHz represents the upper limit for excitation. HFEC above 50 kHz is safe to use for bipolar electrosurgical tissue welding without concerns of excitation propagating beyond the surgical site.

Ureterovesical junction obstruction causes increment in smooth muscle contractility, and cholinergic and adrenergic activity in distal ureter of rabbits

Background/PurposeThe controversy in management of primary obstructed megaureter necessitates further elucidation of the underlying pathophysiology. We evaluated smooth muscle contractility, and cholinergic, adrenergic and serotonergic activity of rabbit distal ureters after ureterovesical junction (UVJ) obstruction. MethodsSham (SH) operation, partial obstruction (PO) and complete obstruction (CO) of the right UVJ were performed in rabbits. Three weeks later, distal ureters were isolated; spontaneous contractions (SC), contractile responses to electrical field stimulation (EFS), high KCl, carbachol, phenylephrine and serotonin were recorded. ResultsSC amplitudes increased in CO compared to PO and SH (p<0.001). SC frequency was higher in CO (p<0.05). EFS-induced contraction amplitudes were greater in CO than other groups (p<0.05). High KCl-induced contractions were greater in CO (p<0.001) and PO (p<0.01). Carbachol-induced contractility was enhanced in CO and PO (p<0.05). Contractile response to phenylephrine was greater in CO than other groups (p<0.05). Serotonin induced contractile responses in CO and PO, greater in CO (p<0.05). UVJ obstruction also increased spontaneous contractility in contralateral PO and CO ureters. ConclusionsUVJ obstruction increased spontaneous and neurotransmitter-induced contractions in an obstruction grade-dependent manner. Obstruction also altered contractility of the contralateral ureters. Our findings may serve to provide further understanding of the pathophysiology of megaureter.

Opposite Effect of Mast Cell Stabilizers Ketotifen and Tranilast on the Vasoconstrictor Response to Electrical Field Stimulation in Rat Mesenteric Artery

ObjectivesWe analyzed whether mast cell stabilization by either ketotifen or tranilast could alter either sympathetic or nitrergic innervation function in rat mesenteric arteries.MethodsElectrical field stimulation (EFS)-induced contraction was analyzed in mesenteric segments from 6-month-old Wistar rats in three experimental groups: control, 3-hour ketotifen incubated (0.1 αmol/L), and 3-hour tranilast incubated (0.1 mmol/L). To assess the possible participation of nitrergic or sympathetic innervation, EFS contraction was analyzed in the presence of non-selective nitric oxide synthase (NOS) inhibitor L-NAME (0.1 mmol/L), α-adrenergic receptor antagonist phentolamine (0.1 µmol/L), or the neurotoxin 6-hydroxydopamine (6-OHDA, 1.46 mmol/L). Nitric oxide (NO) and superoxide anion (O2 .-) levels were measured, as were vasomotor responses to noradrenaline (NA) and to NO donor DEA-NO, in the presence and absence of 0.1 mmol/L tempol. Phosphorylated neuronal NOS (P-nNOS) expression was also analyzed.ResultsEFS-induced contraction was increased by ketotifen and decreased by tranilast. L-NAME increased the vasoconstrictor response to EFS only in control segments. The vasodilator response to DEA-NO was higher in ketotifen- and tranilast-incubated segments, while tempol increased vasodilator response to DEA-NO only in control segments. Both NO and O2 - release, and P-nNOS expression were diminished by ketotifen and by tranilast treatment. The decrease in EFS-induced contraction produced by phentolamine was lower in tranilast-incubated segments. NA vasomotor response was decreased only by tranilast. The remnant vasoconstriction observed in control and ketotifen-incubated segments was abolished by 6-OHDA.ConclusionWhile both ketotifen and tranilast diminish nitrergic innervation function, only tranilast diminishes sympathetic innnervation function, thus they alter the vasoconstrictor response to EFS in opposing manners.

The inhibitory effects of antimuscarinic autoantibodies in the sera of primary Sjogren syndrome patients on the gastrointestinal motility

Impairment of gastrointestinal tract (GI) function, including delayed gastric emptying and colonic dysmotility, are common features of primary Sjögren's syndrome (SS). However, the pathogenesis remains largely unknown. The aim of the current study was to investigate the role of functional autoantibodies to the muscarinic receptor in mediating GI dysfunction associated with primary SS. The effect of SS or normal immunoglobulin G (IgG) on smooth muscle (SM) motility was assessed by comparing the amplitude of carbachol (CCh) or electrical field stimulation (EFS) – induced muscle contraction before and after IgG application. Muscarinic receptor type 3 (M3R) played a dominant role in both colon and gastric SM contraction, while M2R was partly involved in gastric smooth muscle contraction. Preincubation for 1h of the colon and gastric SM strips with 1mg/ml purified IgG from the sera of four primary SS patients (SS IgG) significantly inhibited carbachol-induced smooth muscle contraction (CISC) over a range of CCh concentrations, whereas IgG from healthy controls had little effect. Incubation of the colon SM strips with SS IgG also inhibited EFS-induced colon muscle contraction, which was mimicked by the M3R-selective blocker, 4-DAMP. SR1403330, an NK1 antagonist, had little effect on EFS-mediated colonic SM contraction. The results suggest that autoantibodies isolated from primary SS patients’ sera inhibit muscarinic receptor-mediated cholinergic neurotransmission in mouse colon and stomach, which may provide clues for explaining the GI dysfunction seen in patients with primary SS.

Lubiprostone Increases Small Intestinal Smooth Muscle Contractions Through a Prostaglandin E Receptor 1 (EP)-mediated Pathway

Background/AimsLubiprostone, a chloride channel type 2 (ClC-2) activator, was thought to treat constipation by enhancing intestinal secretion. It has been associated with increased intestinal transit and delayed gastric emptying. Structurally similar to prostones with up to 54% prostaglandin E2 activity on prostaglandin E receptor 1 (EP1), lubiprostone may also exert EP1-mediated procontractile effect on intestinal smooth muscles. We investigated lubiprostone's effects on intestinal smooth muscle contractions and pyloric sphincter tone.MethodsIsolated murine small intestinal (longitudinal and circular) and pyloric tissues were mounted in organ baths with modified Krebs solution for isometric recording. Basal muscle tension and response to electrical field stimulation (EFS; 2 ms pulses/10 V/6 Hz/30 sec train) were measured with lubiprostone (10-10-10-5 M) ± EP1 antagonist. Significance was established using Student t test and P < 0.05.ResultsLubiprostone had no effect on the basal tension or EFS-induced contractions of longitudinal muscles. With circular muscles, lubiprostone caused a dose-dependent increase in EFS-induced contractions (2.11 ± 0.88 to 4.43 ± 1.38 N/g, P = 0.020) that was inhibited by pretreatment with EP1 antagonist (1.69 ± 0.70 vs. 4.43 ± 1.38 N/g, P = 0.030). Lubiprostone had no effect on circular muscle basal tension, but it induced a dose-dependent increase in pyloric basal tone (1.07 ± 0.01 to 1.97 ± 0.86 fold increase, P < 0.05) that was inhibited by EP1 antagonist.ConclusionsIn mice, lubiprostone caused a dose-dependent and EP1-mediated increase in contractility of circular but not longitudinal small intestinal smooth muscles, and in basal tone of the pylorus. These findings suggest another mechanism for lubiprostone's observed clinical effects on gastrointestinal motility.

Aerobic exercise training increases neuronal nitric oxide release and bioavailability and decreases noradrenaline release in mesenteric artery from spontaneously hypertensive rats

To study the effect of aerobic exercise training on sympathetic, nitrergic and sensory innervation function in superior mesenteric artery from spontaneously hypertensive rats (SHRs). De-endothelized vascular rings from sedentary and trained SHRs (treadmill 12 weeks) were used. Vasomotor responses to electrical field stimulation (EFS), noradrenaline, nitric oxide donor DEA-NO and calcitonin gene-related peptide (CGRP) were studied. Neuronal nitric oxide synthase (nNOS) expression and nitric oxide, superoxide anions (O(2.-)), noradrenaline and CGRP levels were also determined. Aerobic exercise training decreased vasoconstrictor response to EFS but increased noradrenaline response. Phentolamine decreased while N(ω)-nitro-(L)-arginine methyl ester ((L)-NAME) increased the response to EFS; the effect of both drugs was greater in trained animals. Training also decreased noradrenaline release and O(2.-) production and increased nNOS expression, nitric oxide release and the vasodilator response to DEA-NO. The O(2.-) scavenger tempol increased DEA-NO-induced vasodilation only in sedentary rats. The EFS-induced contraction was increased to a similar extent in both experimental groups by preincubation with CGRP (8-37). CGRP release and vasodilator response were not modified by training. Aerobic exercise training decreases contractile response to EFS in mesenteric artery from SHRs. This effect is the net result of decreased noradrenaline release, increased sensitivity to the vasoconstrictive effects of noradrenaline and increased neuronal nitric oxide release and bioavailability. These modifications might contribute to the beneficial effects of aerobic exercise training on blood pressure.

Functional BK channels facilitate the β3-adrenoceptor agonist-mediated relaxation of nerve-evoked contractions in rat urinary bladder smooth muscle isolated strips

The large-conductance voltage- and Ca2+-activated K+ (BK) channel is a major regulator of detrusor smooth muscle (DSM) contractility thus facilitating urinary bladder function. Recent findings suggest that activation of β3-adrenoceptors causes DSM relaxation. However, it is unknown whether the β3-adrenoceptor-mediated DSM relaxation is BK channel-dependent during nerve-evoked contractions. To test this hypothesis, we induced nerve-evoked contractions in rat DSM isolated strips by using a tissue bath system equipped with platinum electrodes for electrical field stimulation (EFS). (±)-(R*,R*)-[4-[2-[[2-(3-chlorophenyl)-2-hydroxyethyl]amino]propyl]phenoxy] acetic acid sodium hydrate (BRL37344), a β3-adrenoceptor agonist, significantly decreased the amplitude and muscle force of the 20Hz EFS-induced DSM contractions in a concentration-dependent manner. The BRL37344 inhibitory effect was significantly antagonized by 1-(2-ethylphenoxy)-3-[[(1S)-1,2,3,4-tetrahydro-1-naphthalenyl]amino]-(2S)-2-propanol hydrochloride (SR59230A), a β3-adrenoceptor antagonist. We further isolated the cholinergic from the purinergic component of the 0.5–50Hz EFS-induced DSM contractions by using selective inhibitors, atropine as well as suramin and α,β-methylene-ATP. We found that BRL37344 inhibited both the purinergic and cholinergic components of the nerve-evoked contractions in rat DSM isolated strips. The pharmacological blockade of the BK channels with iberiotoxin, a selective BK channel inhibitor, increased the amplitude and muscle force of the 20Hz EFS-induced contractions in rat DSM isolated strips. In the presence of iberiotoxin, there was a significant reduction in the BRL37344-induced inhibition of the 20Hz EFS-induced contractions in rat DSM isolated strips. These latter findings suggest that BK channels play a critical role in the β3-adrenoceptor-mediated inhibition of rat DSM nerve-evoked contractions.

Alteration of neuromuscular transmissions in the hamster colon following the resolution of TNBS-induced colitis

The aim of this study was to determine whether trinitrobenzene sulfonic acid-induced colitis leads to alterations in enteric neuronal transmission in hamsters. We assessed the mechanical responses induced by the application of electrical field stimulation (EFS) in isolated segments of the distal colon. The EFS-induced relaxation and contraction were blocked by a nitric oxide synthase inhibitor and by the combination of antagonists for tachykinin NK1 and NK2 receptors and muscarinic acetylcholine receptors, respectively. The mechanical responses to EFS were attenuated in the inflamed colon at 7days and were recovered by 30days after inflammation treatment. In addition, we found that purinergic and opioidergic excitatory neural components are expressed following the resolution of colitis. These results suggest that colonic inflammation causes indiscriminate damage to enteric neurons but that neuronal components are restored and that new excitatory neural components, compensating for the contractile responses in smooth muscle after colitis, are expressed.

The effects of α-defensin 1 on electrical field stimulation-induced contraction of rat bladders

This study was designed to investigate the effects of α-defensin 1 on electrical field stimulation (EFS)-induced contractions in isolated rat bladder detrusor muscles. We evaluated the effects of α-defensin 1 (50pM∼5nM) on EFS-induced contractions in the detrusor smooth muscles from 35 rats (2–30Hz). Bladder strips were pretreated with α-defensin 1 and then changes of contractions by adenosine 5′-triphosphate (ATP) were observed. Moreover, after pretreatment with α-defensin 1 for 10min, changes in concentration-response curves to hydrogen peroxide (H2O2) were investigated. Alpha-defensin 1 has increased EFS-induced contractions, significantly, and the contractile response to ATP (1,2,5,10mM) was also increased significantly when strips were pretreated with α-defensin 1. In addition, alpha-defensin 1 increased H2O2-induced contractions. The present study demonstrates that α-defensin 1 increases EFS-induced contractions of rat detrusor muscles via purinergic contraction coupled with the Rho kinase pathway.

38 MOUSE VOIDING PATTERNS ARE STRAIN-SPECIFIC: A NOVEL ASSAY FOR AGE-RELATED DEVELOPMENT OF LOWER URINARY TRACT SYMPTOMS (LUTS)

They were divided into two groups; BOO and sham operated (SHAM) groups. BOO was created by a partial ligation of the urethra with a nylon suture, whereas SHAM group of rats underwent only exposure of the urethra without ligation. Six weeks after surgery, three experiments were performed: 1) mRNA expression of N-Ca in the bladder, 2) in vitro functional studies of detrusor strips focusing on the effects of -conotoxin GVIA (CTX; a selevctive N-Ca inhibitor, 10 6 M) on carbachol (CCh)-induced and electrical field stimulation (EFS)-induced contractions, and 3) cystometric investiogations (CMG) with continuous salineinstillation in a conscious state with intravenous administration of CTX (3 g/kg). RESULTS: N-Ca mRNA expression in the detrusor was about 50 times higher in BOO than SHAM group. CTX did not suppress CCh-induced contractions in either group. The EFS-induced contractions were weaker in the detrusor of BOO than SHAM group. CTX significantly suppressed the EFS-induced contractions in BOO group, but not in SHAM group (Figure 1 A, B). The suppressive effect of CTX on the EFS-induced contractions was more pronounced in the cholinergic component at 10 Hz. In CMG, the number of non-voiding contractions (NVCs) was significantly greater in BOO group than SHAM group. The number of NVCs was significantly decreased after CTXadministration in BOO rats (Figure 1 C). CONCLUSIONS: The results of the current study suggest that N-Ca may have an important role in facilitating release of excitatory transmitters including ACh from the efferent nerve terminals innervating the detrusor of the BOO rats, and this may link to the development of detrusor overactivity associated with BOO.

1595 INHIBITION OF EFS-, PHENYLEPHRINE-, AND THROMBOXANE-INDUCED CONTRACTION BY THE RECEPTOR ANTAGONIST, PICOTAMIDE, IN HUMAN PROSTATE SMOOTH MUSCLE

You have accessJournal of UrologyBenign Prostatic Hyperplasia: Basic Research1 Apr 20131595 INHIBITION OF EFS-, PHENYLEPHRINE-, AND THROMBOXANE-INDUCED CONTRACTION BY THE RECEPTOR ANTAGONIST, PICOTAMIDE, IN HUMAN PROSTATE SMOOTH MUSCLE Martin Hennenberg, Marijan Miljak, Frank Strittmatter, Yasemin Hocaoglu, Beata Rutz, Claudius Füllhase, Christian G. Stief, and Christian Gratzke Martin HennenbergMartin Hennenberg Munich, Germany More articles by this author , Marijan MiljakMarijan Miljak Munich, Germany More articles by this author , Frank StrittmatterFrank Strittmatter Munich, Germany More articles by this author , Yasemin HocaogluYasemin Hocaoglu Munich, Germany More articles by this author , Beata RutzBeata Rutz Munich, Germany More articles by this author , Claudius FüllhaseClaudius Füllhase Munich, Germany More articles by this author , Christian G. StiefChristian G. Stief Munich, Germany More articles by this author , and Christian GratzkeChristian Gratzke Munich, Germany More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2013.02.3145AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Inhibition of prostate smooth muscle contraction is an important strategy for medical treatment of lower urinary tract symptoms (LUTS). Besides α1-adrenoceptors, prostate smooth muscle contraction can be induced by activation of thromboxane (TXA2) receptors. However, effects of TXA2 receptor (TXA2R) antagonists on contraction have not been tested in the lower urinary tract to date. Objective: To examine the effects of TXA2R antagonists on contraction of human prostate tissue. METHODS Prostate tissues were obtained from radical prostatectomy. The effects of picotamide (300 μM), L-665,240 (3 μM), and seratrodast (3 μM) on electric field stimulation- (EFS-), phenylephrine-, and U46619-induced contractions were studied in the organ bath. Phenylephrine is an α1-adrenoceptor agonist. U46619 is a stable thromboxane analogue, activating the TXA2 receptor. Expression of TXA2R and TXA2 synthase (TXS) was examined by fluorescence stainings. RESULTS EFS (2-32 Hz) induced frequency-dependent contractions of human prostate strips, which were almost abolished by picotamide (p<0.0004 at all frequencies), but not by vehicle (DMSO) (n=10 patients). Phenylephrine (0.1-100 μM) and U46619 (0.1-30 μM) induced concentration-dependent contractions of prostate strips, which were significantly blocked by picotamide (p=0.00017-p=0.047 for phenylephrine, p=0.019-p=0.042 for U46619), but not by vehicle (n=6-10 patients). In contrast, L-665,240 or seratrodast were without effects on EFS-induced contractions. Immunoreactivity for TXA2R was observed in the stroma, and in the epithel of glands. TXA2R immunoreactivity colocalized with immunoreactivities of the smooth muscle markers, α-smooth muscle actin (αSMA) and calponin, with the epithelial markers, pan-cytokeratin and prostate-specific antigen (PSA), and with TXS. Immunoreactivity for TXS was observed in the stroma and the epithel. TXS immunoreactivity colocalized with calponin, and pan-cytokeratin. CONCLUSIONS The receptor antagonist picotamide inhibits α1-adrenergic, TXA2-mediated, and EFS-induced contractions in the human prostate. To the best of our knowledge, this is the first antagonist showing simultaneous interventions into different contraction systems of the prostate. In vivo studies are mandatory to examine urodynamic effects of picotamide, and to assess its efficacy for the treatment of LUTS. © 2013 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 189Issue 4SApril 2013Page: e655-e656 Advertisement Copyright & Permissions© 2013 by American Urological Association Education and Research, Inc.MetricsAuthor Information Martin Hennenberg Munich, Germany More articles by this author Marijan Miljak Munich, Germany More articles by this author Frank Strittmatter Munich, Germany More articles by this author Yasemin Hocaoglu Munich, Germany More articles by this author Beata Rutz Munich, Germany More articles by this author Claudius Füllhase Munich, Germany More articles by this author Christian G. Stief Munich, Germany More articles by this author Christian Gratzke Munich, Germany More articles by this author Expand All Advertisement Advertisement PDF DownloadLoading ...

1596 THE CAMP EFFECTOR EPAC ACTIVATES ELK1 TRANSCRIPTION FACTOR IN HUMAN PROSTATE SMOOTH MUSCLE, AND IS A MINOR REGULATOR OF α1-ADRENERGIC CONTRACTION

lial markers, pan-cytokeratin and prostate-specific antigen (PSA), and with TXS. Immunoreactivity for TXS was observed in the stroma and the epithel. TXS immunoreactivity colocalized with calponin, and pancytokeratin. CONCLUSIONS: The receptor antagonist picotamide inhibits 1-adrenergic, TXA2-mediated, and EFS-induced contractions in the human prostate. To the best of our knowledge, this is the first antagonist showing simultaneous interventions into different contraction systems of the prostate. In vivo studies are mandatory to examine urodynamic effects of picotamide, and to assess its efficacy for the treatment of LUTS.

289 UPREGULATION OF CAVEOLIN PROTEINS BY ANGIOTENSIN II ENHANCES DETRUSOR CONTRACTION

INTRODUCTION AND OBJECTIVES: Bladder smooth muscle (BSM) cells release Angiotensin II (AngII) in response to mechanical stress and the angiotensin system is upregulated with bladder obstruction. AngII-mediated bladder contractions require intact caveolae, membrane microdomains involved in signal transduction regulation. These microdomains were found to be altered in hypertrophic detrusor muscle from obstructed bladders. This study investigated the effect of AngII on caveolin expression and whether changes in caveolins induced by AngII are associated with functional alterations. METHODS: Rat bladder tissue without mucosa was challenged for 8 hours with exogenous AngII, or with continuous electrical field stimulation (EFS) in the presence or absence of the AngII receptor-1 (AT1R) antagonist losartan (los). Changes in amplitude of contractions in response to treatments were recorded during the 8 hours. The effect of EFS on the release of endogenous AngII was determined by enzyme immunoassay. After stimulation, caveolin gene and protein expression was determined by real time rt-PCR and western blot. The interaction between AT1R and caveolin proteins was investigated by immunoprecipitation. RESULTS: Exposure of BSM tissue to AngII upregulated Cav-1, Cav-2 and Cav-3 gene and protein expression compared to non-stimulated tissue. Caveolin gene expression was similarly upregulated after 8 hours of EFS, but remained unchanged if EFS was delivered in the presence of los. Release of endogenous AngII was increased in EFS stimulated tissue compared to control. Continuous AngII exposure increased the amplitude of spontaneous activity compared to non-stimulated tissue. In addition, the amplitude of EFSinduced contractions was augmented after 8 hours. However, the increase in EFS-induced contractions was prevented by los. AT1R co-precipitated with Cav-1, Cav-2 and Cav-3. CONCLUSIONS: The caveolin protein-AT1R interaction is consistent with the localization of AT1R in caveolae and its regulation by caveolin proteins. The effect of AT1R inhibition on caveolin expression during mechanical perturbations indicates that autocrine/paracrine AngII positively regulates caveolin expression. These findings suggest that endogenous AngII released by BSM initiates a positive feedback response in which AT1R-activated detrusor contractions that are facilitated by caveolae become augmented by an enhanced caveolin-AT1R interaction. These findings may have important implications for pathophysiologic processes associated with altered AngII secretion such as the hypertrophic response to bladder obstruction.

1594 MECHANISM OF DYNAMIC OBSTRUCTION IN BENIGN PROSTATIC HYPERPLASIA: ANALYSES IN RABBIT PROSTATE

You have accessJournal of UrologyBenign Prostatic Hyperplasia: Basic Research1 Apr 20131594 MECHANISM OF DYNAMIC OBSTRUCTION IN BENIGN PROSTATIC HYPERPLASIA: ANALYSES IN RABBIT PROSTATE Soichiro Ogawa, Junya Hata, Ken Kumagai, Norio Takahashi, Nobuhiro Haga, Nobuhiro Kushida, and Yoshiyuki Kojima Soichiro OgawaSoichiro Ogawa Fukushima, Japan More articles by this author , Junya HataJunya Hata Fukushima, Japan More articles by this author , Ken KumagaiKen Kumagai Fukushima, Japan More articles by this author , Norio TakahashiNorio Takahashi Fukushima, Japan More articles by this author , Nobuhiro HagaNobuhiro Haga Fukushima, Japan More articles by this author , Nobuhiro KushidaNobuhiro Kushida Fukushima, Japan More articles by this author , and Yoshiyuki KojimaYoshiyuki Kojima Fukushima, Japan More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2013.02.3144AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES It is known that noradrenaline (NA) induces prostatic smooth muscle contraction. Meanwhile, nitric oxide (NO) relaxes the prostatic smooth muscle. Thus, the balance between the levels of NA and NO determines the tone of prostatic smooth muscle. If the NA-NO balance is poor, according to our hypothesis, the muscle tone becomes stronger, leading to bladder outlet obstruction observed in patients with benign prostatic hyperplasia (BPH). Therefore, we investigated whether aging alters adrenergic and nitrinergic neurotransmission in the prostate. METHODS We used 3-month-old and 2-year-old male rabbits. All animal procedures were approved by the Animal Care Committee of Fukushima Medical University. We performed organ bath study to examine the nerve-mediated contraction. Then, NA and NO release was measured using the microdialysis technique. P < 0.05 was regarded as statistically significant. RESULTS EFS-induced contraction of prostatic strips from the old rabbits was significantly greater than that from the young rabbits (Fig. 1). The dose-response curves for exogenous NA and sodium nitroprusside (SNP), a NO donor, did not show any significant difference between the two groups (Fig. 2). By the microdialysis technique, there was a significant increase in NA release and a decrease in NO release (Fig. 3). CONCLUSIONS Our findings showed that, as a result of aging, NA increased while NO decreased. Furthermore, these results can contribute to a significant increase in the contraction of the old prostate to nerve stimulation. These results appear to show that the use of phosphodiesterase inhibitors in combination with α1-adrenoceptor antagonists is a promising pharmacotherapy for men with BPH. © 2013 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 189Issue 4SApril 2013Page: e655 Advertisement Copyright & Permissions© 2013 by American Urological Association Education and Research, Inc.MetricsAuthor Information Soichiro Ogawa Fukushima, Japan More articles by this author Junya Hata Fukushima, Japan More articles by this author Ken Kumagai Fukushima, Japan More articles by this author Norio Takahashi Fukushima, Japan More articles by this author Nobuhiro Haga Fukushima, Japan More articles by this author Nobuhiro Kushida Fukushima, Japan More articles by this author Yoshiyuki Kojima Fukushima, Japan More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

1604 CONTRACTIONS OF PROSTATES FROM SPONTANEOUSLY HYPERTENSIVE RATS ARE EFFICIENTLY INHIBITED BY SILODOSIN

You have accessJournal of UrologyBenign Prostatic Hyperplasia: Basic Research1 Apr 20131604 CONTRACTIONS OF PROSTATES FROM SPONTANEOUSLY HYPERTENSIVE RATS ARE EFFICIENTLY INHIBITED BY SILODOSIN Roberta Buono, Paolo Dell'Oglio, Giovanni La Croce, Fabio Benigni, Francesco Montorsi, and Petter Hedlund Roberta BuonoRoberta Buono MILAN, Italy More articles by this author , Paolo Dell'OglioPaolo Dell'Oglio MILAN, Italy More articles by this author , Giovanni La CroceGiovanni La Croce MILAN, Italy More articles by this author , Fabio BenigniFabio Benigni MILAN, Italy More articles by this author , Francesco MontorsiFrancesco Montorsi MILAN, Italy More articles by this author , and Petter HedlundPetter Hedlund Linkoping, Sweden More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2013.02.3154AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Silodosin, a highly selective α1A- AR antagonist improves lower urinary tract symptoms (LUTS) in benign prostatic hyperplasia (BPH). Hypertension is a risk factor for BPH and LUTS. Silodosin reduce bladder dysfunction in spontaneously hypertensive rats (SHR). It is not known if silodosin has similar effect on prostate contractility in individuals with or without hypertension. We therefore compared the effects of silodosin, tamsulosin (moderate α1A and α1D selective AR antagonist) and Y27632 (Rho-kinase inhibitor) on nerve-induced contractions of prostates from SHR and Wistar-Kyoto (WKY) controls without hypertension. METHODS After ethical approval, prostate preparations from 4 month old SHR (n=10) and WKY (n=9) rats were functionally evaluated in organbaths. A Grass Polygraph and a Biopac system recorded isometric tension. Nerves were transmurally activated by electrical field stimulation (EFS) with a Grass S48 stimulator (20Hz). Concentration-response curves (0.1-100μM) for silodosin, tamsulosin, or Y27632 were determined. The pIC50 values were determined by linear interpolation. An ANOVA was used for statistical comparisons. RESULTS Resting tensions (RT), responses to 60mM potassium (K), and contractions to EFS were similar for prostate preparations from all rats. For WKY, RT, K and EFS were 1.2±0.1, 0.21±0.03, and 0.10±0.02 mN. Corresponding values were 1.4±0.1, 0.22±0.03, and 0.12±0.02 mN for SHR. All the investigated drugs concentration-dependently inhibited EFS-induced contractions of prostate preparations from WKY and SHR with maximal inhibitory activities (Emax) at 100μM. For silodosin, Emax was 76±8% (p<0.05 vs Y27632) and 94±8% (p<0.05 vs tamsulosin) in prostate preparations from WKY and SHR, respectively. In comparison, Emax for tamsulosin was 44±8% in WKY and 42±5% in SHR (both p<0.05 vs silodosin and Y27632). Y27632 reduced EFS contractions by 90±9% (p<0.05 vs silodosin and tamsulosin) in WKY and 93±5% in SHR (p<0.05 vs WKY). The pIC50 values were similar for the three drugs in prostate preparations from WKY. In SHR prostate preparations, the pIC50 were 5.4±0.3 (silodosin), 5.3±0.2 (Y27632), and >4 (tamsulosin; p<0.05). CONCLUSIONS Silodosin and Y27632 have equal potency and efficacy to inhibit contractions of SHR prostate preparations. Tamsulosin is less active. Considering the uroselectivity of silodosin, the current data may suggest that this α1-AR antagonist could be a preferred choice in the treatment of BPH LUTS in patients with hypertension. © 2013 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 189Issue 4SApril 2013Page: e659 Advertisement Copyright & Permissions© 2013 by American Urological Association Education and Research, Inc.MetricsAuthor Information Roberta Buono MILAN, Italy More articles by this author Paolo Dell'Oglio MILAN, Italy More articles by this author Giovanni La Croce MILAN, Italy More articles by this author Fabio Benigni MILAN, Italy More articles by this author Francesco Montorsi MILAN, Italy More articles by this author Petter Hedlund Linkoping, Sweden More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Effects of the 5-HT4 receptor agonist, cisapride, on neuronally evoked responses in human bladder, urethra, and ileum

This study evaluated the effects of a 5-HT4 agonist, cisapride, on neuronally evoked smooth muscle responses in bladder, urethra and ileum and compared these effects with those of an acetylcholinesterase inhibitor, distigmine. Electrical field stimulation (EFS) was applied to human bladder and ileum smooth muscle strips from human organ transplant donors and to urethral strips from prostatectomy patients, to evoke neuronally mediated smooth muscle responses. EFS induced contractions in bladder and mixed responses, consisting of contractions and relaxations, in urethra and ileum. Relaxations were mediated by nitric oxide while contractions were partially cholinergic (i.e. atropine sensitive). This atropine sensitive component amounted to~95% in bladder and ~75% in ileum, and it was enhanced by distigmine in a concentration dependent manner (0.1-3 μM; ~100-600% increase in bladder and ~50-250% increase in ileum). Cisapride (0.0003-1 μM) also enhanced bladder contractions (~75-100% increase) but had no effect on urethral contractions or relaxations, and modestly enhanced ileum contractions (~10-40% increase). Facilitatory effects of cisapride were reversed by the specific 5-HT4 receptor antagonist, SB-203186 (3 μM), but were resistant to repeated washing in the bladder. These data indicate that 5-HT4 receptor agonists enhanced EFS-induced contractions in bladder and ileum without an effect on urethra and suggest that it may be possible to enhance bladder activity with a dose of cisapride that is at, or below, those producing gastrointestinal (GI) effects. Although distigmine's maximal facilitation of bladder and GI tract function was greater than that of cisapride, at clinically relevant concentrations cisapride showed much greater efficacy.

Imidazoline versus alpha2-adrenoceptors in the control of gastric motility in mice

Several lines of evidence suggest that imidazoline receptors mediate various physiological processes. It is rather difficult, however, to distinguish the imidazoline receptor-mediated effects from the alpha2-adrenoceptor-mediated ones due to the reasonable affinity of most imidazoline ligands for the alpha2-adrenoceptors. In the present study the effects of different imidazoline ligands were tested on the electrical field stimulation (EFS)-induced gastric contractions in wild-type (WT), alpha2A-, alpha2B- and alpha2C-adrenoceptor knockout (KO) mice in order to analyze, whether imidazoline I1 and I2 receptors take part in the regulation of gastric motor activity. Clonidine, moxonidine and rilmenidine inhibited the EFS-induced gastric contractions in a concentration dependent manner in WT, alpha2B- and alpha2C-adrenoceptor KO mice, whereas they had no or only weak effect in alpha2A-adrenoceptor KO mice. Their effects in WT mice were inhibited by idazoxan and BRL 44408, but not by ARC 239, AGN 192403 and BU 224. The endogenous imidazoline receptor ligand agmatine failed to affect the EFS-induced contractions, while harmane (an other endogenous imidazoline receptor ligand) and 2-BFI (a selective imidazoline I2 receptor agonist) exerted a slight effect in both WT and alpha2A-adrenoceptor KO mice, but this was not reversible by idazoxan, AGN 192403 and BU 224. It can be concluded, that the inhibitory effect of the tested imidazoline compounds on cholinergic gastric contractions is mediated mainly by alpha2A-adrenoceptors. Although at higher concentrations other receptors may also contribute to their effects, the lack of inhibition by AGN 192403 and BU 224 suggests that these are not imidazoline I1 and I2 receptors.