Spontaneous Phasic Contractions Research Articles

Urothelium-Derived Prostanoids Enhance Contractility of Urinary Bladder Smooth Muscle and Stimulate Bladder Afferent Nerve Activity in the Mouse.

The transitional epithelial cells (urothelium) that line the lumen of the urinary bladder form a barrier between potentially harmful pathogens, toxins, and other bladder contents and the inner layers of the bladder wall. The urothelium, however, is not simply a passive barrier, as it can produce signaling factors, such as ATP, nitric oxide, prostaglandins and other prostanoids, that can modulate bladder function. We investigated whether select substances produced by the urothelium could directly modulate the contractility of the underlying urinary bladder smooth muscle. Force was measured in isolated strips of mouse urinary bladder with the urothelium intact or denuded. Bladder strips developed spontaneous tone and phasic contractions. In urothelium-intact strips, basal tone, as well as the frequency and amplitude of phasic contractions, were 25%, 32%, and 338% higher than in urothelium-denuded strips, respectively. Basal tone and phasic contractility in urothelium-intact bladder strips were abolished by the cyclooxygenase (COX) inhibitor indomethacin (10 mM) or the voltage-dependent Ca2+ channel blocker diltiazem (50 mM), whereas blocking neuronal sodium channels with tetrodotoxin (1 mM) had no effect. These results suggest that prostanoids produced in the urothelium enhance smooth muscle tone and phasic contractions by activating voltage-dependent Ca2+ channels in the underlying bladder smooth muscle. We went on to demonstrate that blocking COX inhibits the generation of transient pressure events in isolated pressurized bladders and greatly attenuates the afferent nerve activity during bladder filling, suggesting that urothelial prostanoids may also play a role in sensory nerve signaling.

Commonality and heterogeneity of pacemaker mechanisms in the male reproductive organs.

During emission, the first phase of ejaculation, smooth muscle in organs of the male reproductive tract (MRT) vigorously contract upon sympathetic nerve excitation to expel semen consisting of sperm and seminal plasma. During inter-ejaculation phases, the epididymis, seminal vesicles and prostate undergo spontaneous phasic contractions (SPCs), this transporting and maintaining the quality of sperm and seminal plasma. Recent studies have revealed platelet-derived growth factor receptor α-expressing (PDGFRα+) subepithelial interstitial cells in seminal vesicles subserve the role of pacemaker cells that electrically drive SPCs in this organ. PDGFRα+ smooth muscle cells in the epididymis also appear to function as pacemaker cells implicating PDGFRα as a potential signature molecule in MRT pacemaking. The dominant mechanism driving pacemaking in these organs is the cytosolic Ca2+ oscillator. This operates through entrainment of the release-refill cycle of Ca2+ stores, the released Ca2+ ions opening Ca2+-activated chloride channels, including in some cases ANO1 (TMEM16A), with the resultant pacemaker potential activating L-type voltage-dependent Ca2+ channels in the smooth muscle causing contraction (viz. SPCs). A second pacemaker mechanism, namely the membrane oscillator also has a role in specific cases. Further investigations into the commonality and heterogeneity of MRT pacemakers will open an avenue for understanding the pathogenesis of male infertility associated with deterioration of seminal plasma.

Involvement of ANO1 currents in pacemaking of PDGFRα-positive specialised smooth muscle cells in rat caudal epididymis.

The epididymal duct exhibits spontaneous phasic contractions (SPCs) to store and transport sperm. Here, we explored molecular identification of pacemaker cells driving SPCs in the caudal epididymal duct and also investigated properties of pacemaker currents underlying SPCs focusing on ANO1 Ca2+-activated Cl- channels (CaCCs). Immunohistochemistry was performed to visualise the distribution of platelet-derived growth factor receptor α (PDGFRα)- or ANO1-positive cells in the rat caudal epididymal duct. Perforated whole-cell patch clamp technique was applied to enzymatically isolated epididymal cells, while SPCs were recorded with video edge-tracking technique. Immunohistochemistry revealed the distribution of α-smooth muscle actin (α-SMA)-positive cells co-expressing both PDGFRα and ANO1 in the innermost smooth muscle layer. Approximately one-third of isolated epididymis cells exhibited spontaneous transient inward currents (STICs) at the holding potential -60 mV. The reversal potential for STICs was close to the calculated chloride equivalent potential depending on intracellular Cl- concentrations. Ani9 (3 µM), the ANO1 specific inhibitor, decreased both amplitude and frequency of STICs, while cyclopiazonic acid (CPA, 30 µM), a sarco-/endoplasmic reticulum Ca2+-ATPase (SERCA) inhibitor, abolished STICs. Ani9 (3 or 10 µM) reduced the frequency of SPCs without changing their amplitude. Thus, PDGFRα+, ANO1+ specialised smooth muscle cells (SMCs) appear to function as pacemaker cells to electrically drive epididymal SPCs by generating ANO1-dependnet STICs. STICs arising from spontaneous Ca2+ release from intracellular Ca2+ store and subsequent opening of ANO1 result in depolarisations that spread into adjacent SMCs where L-type voltage-dependent Ca2+ channels are activated to develop SPCs.

Pacemaking in the lymphatic system.

Lymphatic collecting vessels exhibit spontaneous phasic contractions that are critical for lymph propulsion and tissue fluid homeostasis. This rhythmic activity is driven by action potentials conducted across the lymphatic muscle cell (LMC) layer to produce entrained contractions. The contraction frequency of a lymphatic collecting vessel displays exquisite mechanosensitivity, with a dynamic range from <1 to >20 contractions per minute. A myogenic pacemaker mechanism intrinsic to the LMCs was initially postulated to account for pressure-dependent chronotropy. Further interrogation into the cellular constituents of the lymphatic vessel wall identified non-muscle cell populations that shared some characteristics with interstitial cells of Cajal, which have pacemaker functions in the gastrointestinal and lower urinary tracts, thus raising the possibility of a non-muscle cell pacemaker. However, recent genetic knockout studies in mice support LMCs and a myogenic origin of the pacemaker activity. LMCs exhibit stochastic, but pressure-sensitive, sarcoplasmic reticulum calcium release (puffs and waves) from IP3R1 receptors, which couple to the calcium-activated chloride channel Anoctamin 1, causing depolarisation. The resulting electrical activity integrates across the highly coupled lymphatic muscle electrical syncytia through connexin 45 to modulate diastolic depolarisation. However, multiple other cation channels may also contribute to the ionic pacemaking cycle. Upon reaching threshold, a voltage-gated calcium channel-dependent action potential fires, resulting in a nearly synchronous calcium global calcium flash within the LMC layer to drive an entrained contraction. This review summarizes the key ion channels potentially responsible for the pressure-dependent chronotropy of lymphatic collecting vessels and various mechanisms of IP3R1 regulation that could contribute to frequency tuning.

PTHrP attenuates spontaneous contractions in detrusor smooth muscle of the rat bladder by activating spontaneous transient outward potassium currents.

Parathyroid hormone-related protein (PTHrP) released from detrusor smooth muscle (DSM) cells upon bladder distension attenuates spontaneous phasic contractions (SPCs) in DSM and associated afferent firing to facilitate urine storage. Here, we investigate the mechanisms underlying PTHrP-induced inhibition of SPCs, focusing on large-conductance Ca2+-activated K+ channels (BK channels) that play a central role in stabilizing DSM excitability. Perforated patch-clamp techniques were applied to DSM cells of the rat bladder dispersed using collagenase. Isometric tension changes were recorded from DSM strips, while intracellular Ca2+ dynamics were visualized using Cal520AM-loaded DSM bundles. DSM cells developed spontaneous transient outward potassium currents (STOCs) arising from the opening of BK channels. PTHrP (10nM) increased the frequency of STOCs without affecting their amplitude at a holding potential of - 30mV but not - 40mV. PTHrP enlarged depolarization-induced, BK-mediated outward currents at membrane potentials positive to + 20mV in a manner sensitive to iberiotoxin (100nM), the BK channel blocker. The PTHrP-induced increases in BK currents were also prevented by inhibitors of sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) (CPA 10µM), L-type voltage-dependent Ca2+ channel (LVDCC) (nifedipine 3µM) or adenylyl cyclase (SQ22536 100µM). PTHrP had no effect on depolarization-induced LVDCC currents. PTHrP suppressed and slowed SPCs in an iberiotoxin (100nM)-sensitive manner. PTHrP also reduced the number of Ca2+ spikes during each burst of spontaneous Ca2+ transients. In conclusion, PTHrP accelerates STOCs discharge presumably by facilitating SR Ca2+ release which prematurely terminates Ca2+ transient bursts resulting in the attenuation of SPCs.

H2O2 enhances the spontaneous phasic contractions of isolated human-bladder strips via activation of TRPA1 channels on sensory nerves and the release of substance P and PGE2

Several studies have indicated that reactive oxygen species (ROS) can lead to detrusor overactivity (DO), but the underlying mechanisms are not known. Hydrogen dioxide (H2O2) is used commonly to investigate the effects of ROS. In present study, we investigated the effects of H2O2 on phasic spontaneous bladder contractions (SBCs) of isolated human-bladder strips (iHBSs) and the underlying mechanisms. Samples of bladder tissue were obtained from 26 patients undergoing cystectomy owing to bladder cancer. SBCs of iHBSs were recorded in organ-bath experiments. H2O2 (1μM–10mM) concentration-dependently increased the SBCs of iHBSs. These enhancing effects could be mimicked by an agonist of transient receptor potential (TRP)A1 channels (allyl isothiocyanate) and blocked with an antagonist of TRPA1 channels (HC030031; 10 μM). H2O2 induced enhancing effects also could be attenuated by desensitizing sensory afferents with capsaicin (10 μM), blocking nerve firing with TTX (1 μM), blocking neurokinin effects with NK2 receptor antagonist (SR48968, 10 μM), and blocking PGE2 synthesis with indomethacin (10 μM), respectively. Our study: (i) suggests activation of TRPA1 channels on bladder sensory afferents, and then release of substance P or PGE2 from sensory nerve terminals, contribute to the H2O2-induced enhancing effects on SBCs of iHBSs; (ii) provides insights for the mechanisms underlying ROS leading to DO; (iii) indicates that targeting TRPA1 channels might be the promising strategy against overactive bladder in conditions associated with excessive production of ROS.

Role of PTHrP in attenuating transient pressure rises and associated afferent nerve activity of the rat bladder.

Parathyroid hormone-related protein (PTHrP) released from detrusor smooth muscle (DSM) as the bladder fills acts as an endogenous DSM relaxant to facilitate bladder storage function. Here, the effects of exogenous PTHrP on transient pressure rises (TPRs) in the bladder and associated afferent nerve activity during bladder filling were investigated. In anaesthetized rats, changes in the intravesical pressure were measured while the bladder was gradually filled with saline. Afferent nerve activity was simultaneously recorded from their centrally disconnected left pelvic nerves. In DSM strips, spontaneous and nerve-evoked contractions were isometrically recorded. The distribution of PTHrP receptors (PTHrPRs) in the bladder wall was also examined by fluorescence immunostaining. The bladders in which the contralateral pelvic nerve was also centrally disconnected developed nifedipine, an L-type voltage-dependent Ca2+ channel blocker-sensitive TPRs (< 3mmHg). Intravenous administration of PTHrP suppressed these TPRs and associated bursts of afferent nerve activity. In the bladders with centrally connected contralateral pelvic nerves, atropine, a muscarinic receptor antagonist-sensitive large TPRs (> 3mmHg) developed in the late filling phase. PTHrP diminished the large TPRs and corresponding surges of afferent nerve activity. In DSM strips, bath-applied PTHrP (10nM) suppressed spontaneous phasic contractions, while less affecting nerve-evoked contractions. PTHrPRs were expressed in DSM cells but not in intramural nerve fibers. Thus, PTHrP appears to suppress bladder TPRs and associated afferent nerve activity even under the influence of low degree of parasympathetic neural input during storage phases. Endogenous PTHrP may indirectly attenuate afferent nerve activity by suppressing TPRs to facilitate urinary accommodation.

SKA-31-induced activation of small-conductance calcium-activated potassium channels decreased modulation of detrusor smooth muscle function in a rat model of obesity

Increased excitability and contractility of detrusor smooth muscle (DSM) cells are associated with overactive bladder (OAB), which is often induced by obesity. Small-conductance Ca2+-activated K+ (SK) channels regulate the excitability and contractility of DSM cells. Selective pharmacological activation of SK channels attenuates hyperpolarization and the decreased relaxation effect in DSM cells in obesity-induced OAB. However, additional data are needed to confirm the regulatory effect of SK channels on the function of DSM cells in obesity-related OAB. The tested hypothesis was that activation of SK channels decreases modulation of DSM function in a rat model of obesity-related OAB. Female Sprague Dawley rats were fed a normal diet (ND) or a high-fat diet (HFD), weighed after 12weeks, and subjected to urodynamic study, patch-clamp electrophysiology, and isometric tension recording. The average body weight and incidence of OAB were increased in the HFD group. Patch-clamp studies revealed that pharmacological activation of SK channels with SKA-31 had attenuated hyperpolarization of DSM cells. In addition, isometric tension recordings indicated that SKA-31 decreased relaxation of spontaneous phasic contractions of DSM strips in the HFD group. Attenuated function of SK channels increased the excitability and contractility of DSM cells, which contributed to the occurrence of OAB, suggesting that SK channels are potential therapeutic targets for control of OAB.

PDGFRα+ subepithelial interstitial cells act as a pacemaker to drive smooth muscle of the guinea pig seminal vesicle

AbstractSmooth muscle cells (SMCs) of the guinea pig seminal vesicle (SV) develop spontaneous phasic contractions, Ca2+ flashes and electrical slow waves in a mucosa‐dependent manner, and thus it was envisaged that pacemaker cells reside in the mucosa. Here, we aimed to identify the pacemaker cells in SV mucosa using intracellular microelectrode and fluorescence Ca2+ imaging techniques. Morphological characteristics of the mucosal pacemaker cells were also investigated using focused ion beam/scanning electron microscopy tomography and fluorescence immunohistochemistry. Two populations of mucosal cells developed spontaneous Ca2+ transients and electrical activity, namely basal epithelial cells (BECs) and subepithelial interstitial cells (SICs). Pancytokeratin‐immunoreactive BECs were located on the apical side of the basement membrane (BM) and generated asynchronous, irregular spontaneous Ca2+ transients and spontaneous transient depolarisations (STDs). The spontaneous Ca2+ transients and STDs were not diminished by 10 μM nifedipine but abolished by 10 μM cyclopiazonic acid (CPA). Platelet‐derived growth factor receptor α (PDGFRα)‐immunoreactive SICs were distributed just beneath the basal side of the BM and developed synchronous Ca2+ oscillations and electrical slow waves, which were suppressed by 3 μM nifedipine and abolished by 10 μM CPA. In SV mucosal preparations in which some smooth muscle bundles remained attached, SICs and residual SMCs developed temporally correlated spontaneous Ca2+ transients. Neurobiotin injected into SICs spread not only to neighbouring SICs but also to neighbouring SMCs or vice versa. These results suggest that PDGFRα+ SICs electrotonically drive the spontaneous contractions of SV smooth muscle.Key points In many visceral smooth muscle organs, spontaneous contractions are electrically driven by non‐muscular pacemaker cells. In guinea pig seminal vesicles (SVs), as yet unidentified mucosal cells appear to drive neighbouring smooth muscle cells (SMCs). Two populations of spontaneously active cells are distributed in the SV mucosa. Basal epithelial cells (BECs) generate asynchronous, irregular spontaneous Ca2+ transients and spontaneous transient depolarisations (STDs). In contrast, subepithelial interstitial cells (SICs) develop synchronous Ca2+ oscillations and electrical slow waves. Pancytokeratin‐immunoreactive (IR) BECs are located on the apical side of the basement membrane (BM), while platelet‐derived growth factor receptor α (PDGFRα)‐IR SICs are located on the basal side of the BM. Spontaneous Ca2+ transients in SICs are synchronised with those in SV SMCs. Dye‐coupling between SICs and SMCs suggests that SICs act as pacemaker cells to drive the spontaneous contractions of SV smooth muscle.

Mechanosensitive modulation of peristaltic contractions in the mouse renal pelvis

The renal pelvis develops spontaneous phasic contractions (SPCs) that underlie pyeloureteric peristalsis. Increased urine flow into the renal pelvis mechanically stimulates the contractile machinery within the renal pelvis to facilitate the propagation of peristalsis. Here, the effects of mechanostimulation of the pelvicalyceal junction (PCJ), where SPCs originate from, on the properties of SPCs were investigated. Using the wire myograph, isometric tension changes in tubular preparations of mouse renal pelvis with calyces were circumferentially measured, while mechanostimuli were applied to the PCJ. Immunohistochemistry and intracellular Ca2+ imaging were performed, respectively, to investigate the distribution and functional roles of mechanosensitive TRPV4 channels in the renal pelvis. SPCs periodically originated from PCJ and propagated distally. Mechanostimulation of the PCJ reduced the frequency of SPCs by about 60%, while almost quadrupling their amplitude. Capsaicin (100 nM), an agonist of TRPV1 channels, or calcitonin gene-related peptide (CGRP) (30 nM) also slowed and enlarged SPCs. A prolonged pre-exposure to capsaicin or BIBN4096 (1 μM), a CGRP receptor antagonist, inhibited the mechanostimulation-induced reduction in the SPC frequency, but did not block the increase in SPC amplitude. TRPV4 immunoreactivity was expressed in both atypical (ASMCs) and typical smooth muscle cells (TSMCs). GSK1016790A (100 nM), a TRPV4 agonist, enlarged SPCs independently of TRPV1 or CGRP without increasing the amplitude of spontaneous Ca2+ transients in TSMCs. Thus, mechanostimulation of PCJ appears to activate TRPV1-expressing sensory nerves, releasing CGRP that predominantly reduce the SPC frequency. Activation of TRPV4 may be involved in the mechanosensitive enlargement of SPCs. (247 words).

PDGFRα+ Interstitial Cells are Effector Cells of PACAP Signaling in Mouse and Human Colon.

Background & AimsPlatelet-derived growth factor receptor α (PDGFRα)-positive interstitial cells (PIC) are interposed between enteric nerve fibers and smooth muscle cells (SMCs) in the tunica muscularis of the gastrointestinal tract. PIC have robust expression of small conductance Ca2+ activated K+ channels 3 (SK3 channels) and transduce inhibitory inputs from purinergic and sympathetic nerves in mouse and human colon. We investigated whether PIC also express pituitary adenylate cyclase-activating polypeptide (PACAP) receptors, PAC1 (PAC1R), and are involved in mediating inhibitory regulation of colonic contractions by PACAP in mouse and human colons.MethodsGene expression analysis, Ca2+ imaging, and contractile experiments were performed on mouse colonic muscles. Ca2+ imaging, intracellular electrical recordings, and contractile experiments were performed on human colonic muscles.ResultsAdcyap1r1 (encoding PAC1R) is highly expressed in mouse PIC. Interstitial cells of Cajal (ICC) and SMCs expressed far lower levels of Adcyap1r. Vipr1 and Vipr2 were expressed at low levels in PIC, ICC, and SMCs. PACAP elicited Ca2+ transients in mouse PIC and inhibited spontaneous phasic contractions via SK channels. In human colonic muscles, PAC1R agonists elicited Ca2+ transients in PIC, hyperpolarized SMCs through SK channels and inhibited spontaneous phasic contractions.ConclusionsPIC of mouse and human colon utilize PAC1R-SK channel signal pathway to inhibit colonic contractions in response to PACAP. Effects of PACAP are in addition to the previously described purinergic and sympathetic inputs to PIC. Thus, PIC integrate inhibitory inputs from at least 3 neurotransmitters and utilize several types of receptors to activate SK channels and regulate colonic contractile behaviors.

Augmented KCa2.3 Channel Feedback Regulation of Oxytocin Stimulated Uterine Strips from Nonpregnant Mice.

Uterine contractions prior to 37 weeks gestation can result in preterm labor with significant risk to the infant. Current tocolytic therapies aimed at suppressing premature uterine contractions are largely ineffective and cause serious side effects. Calcium (Ca2+) dependent contractions of uterine smooth muscle are physiologically limited by the opening of membrane potassium (K+) channels. Exploiting such inherent negative feedback mechanisms may offer new strategies to delay labor and reduce risk. Positive modulation of small conductance Ca2+-activated K+ (KCa2.3) channels with cyclohexyl-[2-(3,5-dimethyl-pyrazol-1-yl)-6-methyl-pyrimidin-4-yl]-amine (CyPPA), effectively decreases uterine contractions. This study investigates whether the receptor agonist oxytocin might solicit KCa2.3 channel feedback that facilitates CyPPA suppression of uterine contractions. Using isometric force myography, we found that spontaneous phasic contractions of myometrial tissue from nonpregnant mice were suppressed by CyPPA and, in the presence of CyPPA, oxytocin failed to augment contractions. In tissues exposed to oxytocin, depletion of internal Ca2+ stores with cyclopiazonic acid (CPA) impaired CyPPA relaxation, whereas blockade of nonselective cation channels (NSCC) using gadolinium (Gd3+) had no significant effect. Immunofluorescence revealed close proximity of KCa2.3 channels and ER inositol trisphosphate receptors (IP3Rs) within myometrial smooth muscle cells. The findings suggest internal Ca2+ stores play a role in KCa2.3-dependent feedback control of uterine contraction and offer new insights for tocolytic therapies.

Effect of DA-9701 on the Gastrointestinal Motility in the Streptozotocin-Induced Diabetic Mice.

Background: Compared to the general population, diabetic patients experience more frequent episodes of gastrointestinal (GI) motility dysfunction, owing to the disruption of functional innervations. DA-9701 is a new prokinetic agent formulated from the extracts of Pharbitidis semen and Corydalis tuber. Aim: To investigate the effect of DA-9701 on GI motility in an animal model of streptozotocin (STZ)-induced diabetes. Methods: Diabetes was induced in mice by intraperitoneal injection of STZ (40 mg/kg of body weight in 0.1 M citrate buffer) for 3 days. Diabetic mice were divided into four groups and administered DA-9701 in different doses (1, 3, and 10 mg/kg) or placebo for 2 weeks. Intestinal transit was assessed using charcoal meal movement. GI isometric contraction was measured by applying an isometric force transducer on a circular muscle strip of the antrum, ileum, and proximal colon of sacrificed mice. Gastric emptying rate was evaluated by measuring the dye percentage remaining in the stomach relative to the total dye amount recovered in a standardization group of mice. Results: Body weight and antral and small intestinal motility were less in diabetic mice than in control mice, and colonic motility was similar in both. DA-9701 showed a dose-dependent increase in the amplitude of spontaneous phasic contractions in the antrum, ileum, and colon in diabetic mice without influencing body weight or blood glucose levels. The degree of improvement was comparable between diabetic and control mice. Intestinal transit was significantly more delayed in diabetic mice than in controls (43 ± 7% vs. 67 ± 8%, p < 0.05); however, DA-9701 restored the delayed intestinal transit more effectively compared to placebo (75% vs. 50%). The gastric emptying rate was significantly more delayed in diabetic mice than in controls (43 ± 10% vs. 62 ± 12%, p < 0.05), and was improved by DA-9701 in a dose-dependent manner (50%, 55%, and 60% in mice treated with 1, 3, and 10 mg/kg of DA-9701, respectively, vs. 43% in placebo-treated and 60% in control mice). Conclusions: DA-9701 improved GI contractility without affecting blood sugar and body weight in diabetic mice. DA-9701 could improve the decreased GI motility and clinical symptoms in progressive diabetic patients.

Complementary mechanisms of modulation of spontaneous phasic contractions by the gaseous signalling molecules NO, H2S, HNO and the polysulfide Na2S3 in the rat colon.

Reactive oxygen and nitrogen species may be produced during inflammation leading to the formation of NO, H2S or HNO. Enzymes such as iNOS, CSE and CBS might also be responsible for polysulfide production. Since these signalling molecules might have an impact on colonic motility, the aim of this study was to compare their effect on rat colonic slow phasic contractions (SPC). Organ bath measurements with strips obtained from rat proximal colon were performed using the polysulfide Na2S3, sodium nitroprusside (NaNP), sodium hydrogen sulfide (NaHS), Angeli's salt as NO, H2S, and HNO donors, respectively. TTX (1µM) was used to block neuronal activity. All four molecules, concentration-dependently, inhibited the amplitude and frequency of SPC both in the circular and longitudinal muscle layer. The relative potency was NaNP>Angeli's salt>NaHS>Na2S3. The inhibitory response induced by NaNP (1µM) and Angeli's salt (50µM) was reversed by ODQ (10µM) whereas the inhibitory effect of NaHS (1mM) was reversed by apamin (1µM) and glibenclamide (10µM). Na2S3 (1mM) response was partially reversed by apamin (1µM) and glibenclamide (10µM). High concentrations of Na2S3 caused an increase in tone. Low concentrations of NaHS or Na2S3 did not potentiate NaNP responses. All signalling molecules inhibit SPC in both muscle layers. The effect is independent of neural activity and involves guanylyl cyclase (NO and HNO) and SKCa and KATP channels (NaHS or Na2S3). Other pathways might also be involved in Na2S3 responses. Accordingly, complementary mechanisms of inhibition might be attributable to these signalling molecules.

Mechanisms underlying spontaneous phasic contractions and sympathetic control of smooth muscle in the rat caudal epididymis.

Here we investigate mechanisms underlying spontaneous phasic contractions (SPCs) and sympathetic control of contractility in the rat epididymis, a long tubular duct involved in transportation and maturation of sperm. Longitudinal contractions of short segments (~ 1.5mm) of rat proximal and distal caudal epididymal duct were measured + / - nerve stimulation. The extent of sympathetic innervation of these duct regions was determined by immunohistochemistry. Proximal caudal duct segments (150-300μm dia.) exhibited SPCs, while distal segments (350-500μm) were quiescent in ~ 80% of preparations. SPC amplitude and frequency were reduced by the L-type voltage-dependent Ca2+ channel (LVDCC) blocker nifedipine (1μM), with the T-type voltage-dependent Ca2+ channel (TVDCC) blocker ML218 (1μM) specifically decreasing SPC frequency. SPCs were inhibited upon blockade of the SR/ER Ca2+-ATPase (CPA 10μM). SPCs were also inhibited by caffeine (1μM), 2-APB (100μM), niflumic acid (100μM), or by lowering extracellular [Cl-] from 134.4 to 12.4mM but not by ryanodine (25μM) or tetracaine (100μM). Electrical field stimulation (EFS) at 2Hz for 60s caused a sustained α1-adrenoceptor-sensitive contraction in distal segments and enhanced and/or induced α2-adrenoceptor-sensitive oscillatory phasic contractions in proximal and distal segments, the latter mimicked by application of the α2-adrenoceptor agonist clonidine. We hypothesise that SPCs in the proximal cauda are triggered by pacemaker mechanisms involving rhythmic IP3 receptor-operated SR/ER store Ca2+ release and resultant activation of CaCC with TVDCCs and possibly LVDCCs subserving in this process. Sympathetic nerve-released noradrenaline induces α2-adrenoceptor-mediated phasic contractions in the proximal and distal cauda. These findings provide new pharmacological targets for male infertility and contraception.

The anxiolytic sertraline reduces the impact of psychological stress on bladder function in mice

AimsTo determine if treatment with the selective serotonin reuptake inhibitor (SSRI) sertraline reduces the bladder dysfunction caused by water avoidance stress in mice. Main methodsAdult female mice were randomly allocated to (1) Unstressed, (2) Stressed or (3) Stress + Sertraline experimental groups. Stressed mice were subjected to water avoidance for 1 h/day for 10 days and received sertraline or vehicle in drinking water, starting 10-days prior to the first stress exposure. Age matched control/unstressed mice were house under normal conditions without stress exposure. Voiding behaviour was assessed throughout the experimental protocol. After the final stress exposure, a blood sample was taken to measure plasma corticosterone levels and bladders were removed, catheterised and intravesical pressure responses recorded during distension and in response to pharmacological agents. Key findingsPlasma corticosterone levels in sertraline-treated animals were equivalent to unstressed controls and significantly decreased compared to the stressed group. Voiding frequency was significantly increased in the stressed group, and treatment with sertraline significantly decreased voiding frequency, however, this remained elevated compared to unstressed control animals. Bladders from stressed mice displayed enhanced maximal contractile response to the muscarinic agonist carbachol and greater release of ACh in the serosal fluid, which was reduced to control levels by sertraline treatment. Spontaneous phasic contractions were not altered by stress but were significantly reduced in bladders from sertraline treated animals, relative to controls. SignificanceThese results indicate that management of voiding dysfunction caused by psychological stress may be aided by the addition of an SSRI such as sertraline.

The mast cell activator Compound 48/80 causes phasic urinary bladder smooth muscle contractions independent of histamine release

Inflammatory mediators released from mast cells cause both transient and phasic contractions of urinary bladder smooth muscle (UBSM). These contractions are viewed as initial disruptors of normal bladder function and can lead to profound bladder pathologies. However, the mechanisms responsible for each of these contractions has not been thoroughly investigated. Previously, we discovered that histamine-mediated contractions rapidly desensitize, whereas the mast cell activator compound 48/80 (10 µg/ml) caused both a long-lasting increase in the amplitude and frequency of phasic contractions in UBSM strips as well as a transient increase in baseline tension. Thus, we hypothesized that phasic contractions caused by compound 48/80 depended on the release of histamine from mast cells. Isometric contractility was performed with urothelium-intact UBSM strips from C57BL/6 mice in the presence or absence of the following drugs: the cholinergic antagonist atropine (2 µM); the 5HT2A receptor antagonist MDL 11939 (1 µM); the histamine H1 receptor antagonist fexofenadine (10 µM), the histamine H2 receptor antagonist roxatidine (25 µM), the H2 antagonist cimetidine (10 µM); or the mast cell stabilizer cromolyn (100 µM). None of the compounds tested significantly reduced the amplitude and duration of the spontaneous phasic contractions caused by compound 48/80. However, both MDL 11939 and cromolyn significantly reduced the baseline contraction. Our data suggest that responses to the mast cell activator compound 48/80 are a superimposition of 2 components: (1) a slowly desensitizing increase in baseline tension caused by release of 5-HT from mast cells, and (2) large-amplitude phasic contractions that are histamine- and 5HT-independent. However, the mechanism responsible for increases in the amplitude of phasic contractions remains unclear.

Role of Internal Ca2+ Stores in KCa Channel Feedback Control of Human Uterine Contractions

Background Minute-to-minute regulation of intracellular calcium concentrations is essential to allow a laboring uterus to phasically contract and relax in a manner required for delivery. Cytosolic calcium levels increase through influx of calcium through L-type calcium channels and are amplified by SR calcium release through inositol 1, 4, 5-triphosphate receptor (IP3R) channels and calcium induced calcium release. A specific class of small conductance calcium-activated potassium (KCa2.3) channels has been strongly implicated in the negative feedback control of membrane potential and intracellular calcium levels. Uterine contractions can be suppressed by amplifying this calcium-dependent feedback system. CyPPA, a positive modulator of KCa2.3, reduces contraction amplitudes in human and rodent tissue. Previous studies of endothelial and neuronal cells suggest focused Ca2+ release from internal stores in close proximity to the plasma membrane, may play an important role in KCa channel control. Indeed, close proximity and interaction of endoplasmic reticulum Ca2+ release channels (e.g. IP3Rs) and KCa channels may regulate intracellular calcium in a variety of tissues. This study aims to assess whether internal stores contribute to KCa2.3 dependent feedback regulation of contraction in the human myometrium. Methods Myograph studies were performed on human uterine tissues collected from non-pregnant patients undergoing hysterectomy. Uterine strips were sutured end-to-end to form rings, mounted onto myograph pins, and allowed to develop spontaneous phasic contractions. Concentration-dependent suppression of contractions with the positive KCa2.3 modulator CyPPA (3 μM, 10 μM, 30 μM) was assessed following depletion of internal calcium stores (using the SERCA inhibitor cyclopiazonic acid, CPA) or following treatment with the vehicle DMSO (controls). Results Utilizing myographical recordings and LabChart software, contraction area under curve (AUC) was calculated and normalized to time. Addition of 3 μM CyPPA resulted in a control group mean AUC of 1.287 mN/min (± 0.417) and 2.261 mN/min (± 1.338) for the treatment group [n=5]. The mean AUC with 10 μM CyPPA was 1.129 mN/min (± 0.571) for the control group and 2.164 mN/min (± 1.387) for the treatment group. After 30 μM CyPPA the mean AUC for the control group was 0.734 mN/min (± 0.471) and 1.863 mN/min (± 1.364) for the treatment group. Our group found that CPA pretreated uterine tissue had a significantly higher AUC than the control group (p<0.5), after addition of 30 μM CyPPA. Further analysis demonstrated no significant difference with CyPPA concentrations of 3 and 10 μM, but did reveal a trend of reduced AUC in the control group compared to the treatment group. These findings suggest blocking SERCA and inhibiting SR reuptake of calcium may decrease CyPPAs ability to diminish contractions, especially at higher concentrations. Conclusions Phasic contractions of isolated human uterine strips vary considerably with respect to amplitude and frequency. Overall, the findings suggest internal calcium store release may play an important role in moderating uterine contractions and augmenting KCa2.3 channel suppression of uterine contractility. Extended studies will assess whether this mechanism can be exploited as an effective tocolytic strategy.

Impaired Colonic Contractility and Intestinal Permeability in Symptomatic Uncomplicated Diverticular Disease

Background/AimsImpaired intestinal motility seems to play a crucial role in symptomatic uncomplicated diverticular disease (SUDD), although the mechanism is not clear. The aim of the present study is to explore the contractility patterns of colonic smooth muscle strips (MS) and smooth muscle cells (SMCs) and to assess mucosal integrity in SUDD patients.MethodsMS or SMCs were isolated from specimens of human distal colon of 18 patients undergoing surgery for non-obstructive colonic cancer, among them 9 with SUDD. Spontaneous phasic contractions on strips and morpho-functional parameters on cells were evaluated in basal conditions and in response to acetylcholine (ACh). Mucosal integrity of SUDD colonic biopsies was evaluated by the Ussing Chamber system. Immunohistochemical staining for tight junction protein complex and for Toll-like receptor 4 (TLR4) was performed.ResultsColonic MS of SUDD group showed a significant reduced basal tone and ACh-elicited contraction, compared to the control group (9.5 g and 47.0% in the SUDD group; 14.16 g and 69.0% in the control group; P < 0.05). SMCs of SUDD group showed a maximal contractile response to ACh significantly reduced compared to control group (8.8% vs 16.5%, P < 0.05). SUDD patients displayed lower transepithelial electrical resistance and increased paracellular permeability compared to control group. Immunohistochemical expression of TLR4 was not different in both groups, while tight junction protein complex expression was lower in SUDD patients compared to control group patients.ConclusionIt could be hypothesized that in SUDD, in absence of severe inflammation, an increased intestinal mucosal permeability is related to altered colonic motility probably responsible for symptoms genesis.

Role of PGE2 in colonic motility: PGE2 attenuates spontaneous contractions of circular smooth muscle via EP4 receptors in the rat colon

Colonic motor activity is important for the formation and propulsion of feces. The production of prostaglandins (PGs) in colonic tissue is considered to play a critical role in the generation and regulation of colonic motility. In this study, we investigated the inhibitory effects of PGE2 and selective agonists of four EP receptors on the spontaneous phasic contractions, called ‘giant contractions’ (GCs), of mucosa-free circular smooth muscle strips from the rat middle colon. Neural blockade with tetrodotoxin (TTX) increased the frequency and amplitude of the GCs by about twofold. However, inhibiting PG production with piroxicam reduced the GC frequency in the presence of TTX, but did not affect the GC amplitude. In the presence of both TTX and piroxicam, exogenous PGE2 and each EP receptor agonist were cumulatively added to the tissue bath. In this setting, PGE2, the EP2 agonist ONO-AE1-259, and the EP4 agonist ONO-AE1-329, but not the EP1 agonist ONO-AE-DI-004 or the EP3 agonist ONO-AE-248, concentration-dependently reduced the GC frequency and amplitude. The PGE2-induced inhibition of GC frequency and amplitude was inhibited by the EP4 antagonist ONO-AE3-208, but not by the EP1/2 antagonist AH6809. Immunohistochemistry revealed the EP2 and EP4 receptors were localized in perinuclear sites in circular smooth muscle cells. EP2 immunoreactivity was also located in GFAP-immunoreactive enteroglia, whereas EP4 immunoreactivity was also located in HU (embryonic lethal, abnormal vision [ELAV] protein; a marker of all myenteric neurons)-immunoreactive myenteric nerve cell bodies. These results suggest that the PGs produced in the colonic tissue inhibit the GC frequency and amplitude of circular muscle in the rat middle colon, and is mediated by EP4 receptors expressed in the smooth muscle cells.