Search

Prostate smooth muscle contraction is central in treatment of voiding symptoms in benign prostatic hyperplasia (BPH). Tissues from transurethral resection of the prostate (TURP) and radical prostatectomy (RP) for prostate cancer are widely used to study contractions. However, findings are limited by traumatization in TURP, and uncertain relationship to BPH in RP tissues. This study aims to examine contractions of laser-enucleated tissues. Tissues from holmium/thulium laser enucleation (HoLEP/ThuLEP) and TURP were contracted by KCl, noradrenaline and electric field stimulation (EFS) in an organ bath. Contractions were compared to RP tissues in previous studies. KCl-induced contractions averaged 2.5 mN, 0.7 mN and 3.3 mN in tissues from HoLEP/ThuLEP, TURP and RP, with non-responsive tissues included (2.4% HoLEP/ThuLEP, 37% TURP). Maximum EFS-induced contractions (E max ) averaged 47% of KCl in HoLEP/ThuLEP tissues, 27% in TURP tissues, and 68-235% in 21 previous studies with RP tissues. E max values for noradrenaline averaged 99.7% in HoLEP/ThuLEP tissues, 56% in TURP tissues, and ranged from 92 to 260% in RP tissues. Preoperative α 1 -blocker treatment reduced EFS- and noradrenaline-induced contractions, and increased EC 50 values for noradrenaline in laser-enucleated, catheterized patients, but not in patients without catheterization. Also, the ex vivo application of α 1 -blockers increased the EC 50 values for noradrenaline and reduced E max for EFS. Laser-enucleated tissues allow investigation of prostate smooth muscle contraction in medication-refractory voiding symptoms. Different impacts of preoperative α 1 -blocker treatment on ex vivo contractility in tissues from patients with and without catheterization point to clinically relevant heterogeneity of patients undergoing surgery for BPH., Competing Interests: Declarations. Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Introduction: Carpolobia lutea root extract (CLRE) has been reported to enhance penile erection. However, the mechanism involved is poorly understood. We investigated in vitro mechanisms of CLRE action on contractile activity of rabbit corpus cavernosum (CC)., Methods: Corpus cavernosum strips from four healthy male New Zealand rabbits (2.5-3.0kg) were mounted on an organ chamber and contracted with phenylephrine (PE) (10 -9 to 10 -5 M) and Potassium Chloride (KCl) (10-50mM) before treatment with various concentrations of CLRE (0.1-1.2mg/ml). Interactions between CLRE and a Nitric Oxide Synthase (NOS) inhibitor (N-nitro-l-arginine methyl ester - l-NAME 10 -4 M); guanylyl cyclase inhibitors (Oxalodiazolo 4,3-a quinoxalin-1-one - ODQ 10μM, 20μM, 30μM), and (methylene blue 10-30μM); a cyclooxygenase inhibitor (10 -4 M indomethacin); potassium-channel inhibitors (100μM tetraethyl ammonium TEA), (100ηM apamin) and (glibenclamide 10μM and 20μM); and a calcium-channel inhibitor (-10 -4 M nifedipine) were investigated., Results: Maximal contractions of KCl and PE contracted CC strips were significantly reduced in a concentration-dependent manner (40.8±3.6% and 38.6±4.0% from 64.6±2.9% and 98.1±4.2% respectively). Relaxant effect of CLRE was significantly reduced by ODQ (38.6±4.0% to 6.4±1.3% and 38.6±4.0% to 7.2±1.2%), nifedipine (38.6±4.0% to 21.1±2.7%) and glibenclamide (40.8±3.6% to 31.5±3.3%). However l-NAME, indomethacin, methylene blue, TEA and apamin did not inhibit relaxation by CLRE., Conclusion: Concentration-dependent relaxant effect of CLRE in rabbit CC involves the soluble guanylate cyclase/cyclase Guanosine Monophosphate system, and activation of ATP-dependent K + channels., (Copyright © 2020 Asociación Española de Andrología, Medicina Sexual y Reproductiva. Publicado por Elsevier España, S.L.U. All rights reserved.)

Antispasmodic agents are used for modulating gastrointestinal motility. Several compounds isolated from terrestrial plants have antispasmodic properties. This study aimed to explore the inhibitory effect of the pyrrolidine derivative, asperidine B, isolated from the soil-derived fungus Aspergillus sclerotiorum PSU-RSPG178, on spasmodic activity. Isolated rat ileum was set up in an organ bath. The contractile responses of asperidine B (0.3 to 30 µM) on potassium chloride and acetylcholine-induced contractions were recorded. To investigate its antispasmodic mechanism, CaCl 2 , acetylcholine, Nω-nitro-l-arginine methyl ester (l-NAME), nifedipine, methylene blue and tetraethylammonium chloride (TEA) were tested in the absence or in the presence of asperidine B. Cumulative concentrations of asperidine B reduced the ileal contraction by ~37%. The calcium chloride and acetylcholine-induced ileal contraction was suppressed by asperidine B. The effects of asperidine B combined with nifedipine, atropine or TEA were similar to those treated with nifedipine, atropine or TEA, respectively. In contrast, in the presence of l-NAME and methylene blue, the antispasmodic effect of asperidine B was unaltered. These results suggest that the antispasmodic property of asperidine B is probably due to the blockage of the L-type Ca 2+ channel and is associated with K + channels and muscarinic receptor, possibly by affecting non-selective cation channels and/or releasing intracellular calcium.

Transient receptor potential ankyrin-1 (TRPA1) is a ligand-gated cation channel that responds to endogenous and exogenous irritants. TRPA1 is expressed on multiple cell types throughout the lungs, but previous studies have primarily focused on TRPA1 stimulation of airway sensory nerves. We sought to understand the integrated physiological airway response to TRPA1 stimulation. The TRPA1 agonists allyl isothiocyanate (AITC) and cinnamaldehyde (CINN) were tested in sedated, mechanically ventilated guinea pigs in vivo. Reproducible bronchoconstrictions were induced by electrical stimulation of the vagus nerves. Animals were then treated with intravenous AITC or CINN. AITC and CINN were also tested on isolated guinea pig and mouse tracheas and postmortem human trachealis muscle strips in an organ bath. Tissues were contracted with methacholine, histamine, or potassium chloride and then treated with AITC or CINN. Some airways were pretreated with TRPA1 antagonists, the cyclooxygenase inhibitor indomethacin, the EP 2 receptor antagonist PF 04418948, or tetrodotoxin. AITC and CINN blocked vagally mediated bronchoconstriction in guinea pigs. Pretreatment with indomethacin completely abolished the airway response to TRPA1 agonists. Similarly, AITC and CINN dose-dependently relaxed precontracted guinea pig, mouse, and human airways in the organ bath. AITC- and CINN-induced airway relaxation required TRPA1, prostaglandins, and PGE 2 receptor activation. TRPA1-induced airway relaxation did not require epithelium or tetrodotoxin-sensitive nerves. Finally, AITC blocked airway hyperreactivity in two animal models of allergic asthma. These data demonstrate that stimulation of TRPA1 causes bronchodilation of intact airways and suggest that the TRPA1 pathway is a potential pharmacological target for bronchodilation.

Mesenchymal stem cells (MSCs) were obtained from human bone marrow and amplified in cultures supplemented with human platelet lysate in order to generate myofibroblasts. When MSCs were seeded in solid collagen scaffolds, they differentiated into myofibroblasts that were observed to strongly bind to the substrate, forming a 3D cell scaffold network that developed tension and shortening after KCl stimulation. Moreover, MSC-laden scaffolds recapitulated the Frank-Starling mechanism so that active tension increased in response to increases in the initial length of the contractile system. This constituted a bioengineering tissue that exhibited the contractile properties observed in both striated and smooth muscles. By using the A. F. Huxley formalism, we determined the myosin crossbridge (CB) kinetics of attachment (f1) and detachment (g1 and g2), maximum myosin ATPase activity, molar myosin concentration, unitary CB force and maximum CB efficiency. CB kinetics were dramatically slow, characterizing the non-muscle myosin type IIA (NMMIIA) present in myofibroblasts. When MSCs were seeded in solid collagen scaffolds functionalized with Arg-Gly-Asp (RGD), contractility increased and CB kinetics were modified, whereas the unitary NMMIIA-CB force and maximum CB efficiency did not change. In conclusion, we provided a non-muscle bioengineering tissue whose molecular mechanical characteristics of NMMIIA were very close to those of a non-muscle contractile tissue such as the human placenta., Competing Interests: The authors have declared that no competing interests exist.

In this study we aimed to examine the relaxant effect of berberine, a compound extracted from a variety of herbs, on rat tracheal smooth muscle (TSM) and its possible mechanism(s). Cumulative concentrations of berberine (20, 65, 200 and 600 μg/mL) were added on pre-contracted TSM by methacholine or KCl in non-incubated or incubated tissues with atropine, chlorpheniramine, propranolol, diltiazem, glibenclamide, indomethacin, L-NG-nitro arginine methyl ester (L-NAME) and papaverine. The relaxant effects of theophylline (0.2, 0.4, 0.6 and 0.8 mM) as positive control and saline (1 mL) as negative control were also examined in non-incubated tissues. Berberine showed significant and concentration-dependent relaxant effects in non-incubated tissues contracted by KCl and methacholine (p<0.01 to p<0.001). There was no significant difference in the relaxant effects of berberine between non-incubated and incubated tissues with atropine, propranolol, diltiazem, glibenclamide, and papaverine. The relaxant effects of second concentrations of berberine in incubated tissues with L-NAME, its three lower concentration in incubated tissues with chlorpheniramine and its all concentrations in incubated tissues with indomethacin were significantly lower than non-incubated tissues (p<0.05 to p<0.001). The EC50 values of berberine in incubated tissues with chlorpheniramine was significantly higher than the non-incubated condition (p<0.05). Our findings reveal a relatively potent relaxant effect of berberine that is lower than the effect of theophylline. Proposed mechanisms for the relaxant effect of berberine are histamine (H1) receptor blockade, inhibition of cyclooxygenase pathways and/or nitric oxide formation.

Pelvic organ prolapse, the descent of the pelvic organs from their normal anatomical position, is a common condition among women that is associated with mechanical alterations of the vaginal wall. In order to characterize the complex mechanical behavior of the vagina, we performed planar biaxial tests of vaginal specimens in both the passive (relaxed) and active (contracted) states. Specimens were isolated from virgin, female Long-Evans rats (n = 16) and simultaneously stretched along the longitudinal direction (LD) and circumferential direction (CD) of the vagina. Tissue contraction was induced by electric field stimulation (EFS) at incrementally increasing values of stretch and, subsequently, by KCl. On average, the vagina was stiffer in the CD than in the LD (p < 0.001). The mean maximum EFS-induced active stress was significantly higher in the CD than in the LD (p < 0.01). On the contrary, the mean KCl-induced active stress was lower in the CD than in the LD (p < 0.01). When comparing the mean maximum EFS-induced active stress to the mean KCl-induced active stress, no differences were found in the CD (p = 0.366) but, in the LD, the mean active stress was much higher in response to the KCl stimulation (p < 0.001). Collectively, these results suggest that the anisotropic behavior of the vaginal tissue is determined not only by collagen and smooth muscle fiber organization but also by the innervation.

β-Phenylethylamine (β-PEA) is a trace amine with chemical proximity to biogenic amines and amphetamines. It is an endogenous agonist of trace amine-associated receptors (TAARs) that acts as a neuromodulator of classic neurotransmitters in the central nervous system. At high concentrations, β-PEA contracts smooth muscle, and a role for TAARs in these responses has been postulated. The high dietary intake of trace amines has been associated with such symptoms as hypertension and migraine, especially after the intake of foods containing such compounds. In gastrointestinal tissues, TAAR expression was reported, although the effect of β-PEA on gastric contractile behaviour is unknown. Here, isolated strips that were obtained from the rat gastric fundus were stimulated with high micromolar concentrations of β-PEA. Under resting tonus, β-PEA induced contractions. In contrast, when the strips were previously contracted with KCl, a relaxant response to β-PEA was observed. The contractile effect of β-PEA was inhibited by 5-hydroxytryptamine (5-HT) receptor antagonists (i.e., cyproheptadine and ketanserin) but not by the TAAR 1 antagonist EPPTB. In gastric fundus strips that were previously contracted with 80 mmol/L KCl, the relaxant effect of β-PEA intensified in the presence of 5-HT receptor antagonists, which was inhibited by EPPTB and the adenylyl cyclase inhibitor MDL-12,330A. The guanylyl cyclase inhibitor ODQ did not alter the relaxant effects of β-PEA. In conclusion, β-PEA exerted dual contractile and relaxant effects on rat gastric fundus. The contractile effect appeared to involve the recruitment of 5-HT receptors, and the relaxant effect of β-PEA on KCl-elicited contractions likely involved TAAR 1 ., (© 2018 John Wiley & Sons Australia, Ltd.)

Alpha1-adrenoceptors induce prostate smooth muscle contraction, and hold a prominent role for pathophysiology and therapy of lower urinary tract symptoms in benign prostatic hyperplasia. G protein-coupled receptors are regulated by posttranslational regulation, including phosphorylation by G protein-coupled receptor kinases 2 and 3 (GRK2/3). Although posttranslational adrenoceptor regulation has been recently suggested to occur in the prostate, this is still marginally understood. With the newly developed CMPD101, a small molecule inhibitor with assumed specificity for GRK2/3 is now available. Here, we studied effects of CMPD101 on smooth muscle contraction of human prostate tissue. Electric field stimulation caused frequency-dependent contractions, which were inhibited concentration-dependently by CMPD101 (5 µM, 50 µM). 50 µM of CMPD101 did not affect myosin light chain (MCL) phosphorylation or Rho kinase activity, and did not alter contractions induced by highmolar KCl. Noradrenaline, the α 1 -adrenoceptor agonist phenylephrine, endothelin-1, and the thromboxane A 2 analogue U46619 induced concentration-dependent contractions, which were inhibited by CMPD101 (50 µM). CMPD101 (50 µM) did not change phosphorylation of β 2 -adrenoceptors or β 2 -adrenergic relaxation of prostate strips. Molecular detection by Western blot and peroxidase staining suggested expression of GRK2 and GRK3 in human prostates. Double labeling in fluorescence staining confirmed that immunoreactivity for GRK2 and GRK3 was located to smooth muscle cells in the prostate stroma. In conclusion, CMPD101 inhibits adrenergic, neurogenic, and non-adrenergic smooth muscle contractions in the human prostate. Underlying mechanisms may be independent from GRK inhibition, and from inhibition of MLC kinase and Rho kinase. This may point to unknown properties of CMPD101., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background: Lavandula angustifolia (L. angustifolia) Mill. (Common name Lavender) is used in traditional and folk medicines for the treatment of various diseases including respiratory disorders worldwide. The relaxant effect of the plant on the smooth muscle of some tissues was shown previously. The present study has investigated the role of different receptors and pathways in the relaxant effect of L. angustifolia on tracheal smooth muscle., Methods: Cumulative concentrations of the hydro-ethanolic extract of L. angustifolia flowers (0.5, 1, 2 and 4 mg/ml) were added on pre-contracted tracheal smooth muscle by methacholine (10 μM) or KCl (60 mM) on non-preincubated or preincubated tissues with atropine, chlorpheniramine, propranolol, diltiazem, glibenclamide, indomethacin, ω-nitro-L-arginine methyl ester (L-NAME) and papaverine. The results compared with of theophylline (0.2, 0.4, 0.6 and 0.8 mM) as positive control and saline (1 ml) as negative control., Results: The extract showed concentration-dependent relaxant effects in non-preincubated tracheal smooth muscle contracted by KCl and methacholine (p < 0.05 to p < 0.001). The relaxant effect ofL. angustifolia was not significantly different between non-preincubated and preincubated tissues with chlorpheniramine, propranolol, diltiazem, glibenclamide, and papaverine. However, two higher concentrations of L. angustifolia in preincubated tissues with L-NAME (p < 0.01), indomethacin (p < 0.05 to p < 0.001) and atropine (p < 0.05) showed significantly lower relaxant effects than non-preincubated tissues. The EC 50 values of L. angustifolia in tissues preincubated with indomethacin was significantly higher than non-preincubated trachea (p < 0.05). The effects of three first concentrations of the extract on KCl and methacholine-induced muscle contraction were significantly lower than those of theophylline (p < 0.05 to p < 0.001)., Conclusions: These results indicated a relatively potent relaxant effect ofL. angustifolia that was lower than the effect of theophylline. The possible mechanisms of relaxant effect of this plant on tracheal smooth muscle are muscarinic receptors blockade, inhibition of cyclooxygenase pathways and/or involvement of nitric oxide production. Its clinical applications should be investigated in further studies., (Copyright © 2018 Elsevier Masson SAS. All rights reserved.)

Context: Turmeric is a spice obtained from the root of Curcuma longa L. (Zingiberaceae) with anti-aging, anticancer, anti-Alzheimer's disease, antioxidant and other medicinal properties., Objective: The relaxant effect of C. longa on rat tracheal smooth muscle and its possible mechanisms were investigated in this study., Materials and Methods: The relaxant effects of four cumulative concentrations of hydro-ethanol extract of C. longa (6.25, 12.5, 25, 50 mg/mL) were studied on tracheal smooth muscle precontracted by methacholine or KCl in non-incubated or incubated with different substances including propranolol, diltiazem, L-NAME, glibenclamide, atropine, chlorpheniramine, indomethacin and papaverine. The duration of the study was 84 days., Results: In non-incubated tracheal smooth muscle, the extract of C. longa showed significant concentration-dependent relaxant effects (p < 0.001 for all concentrations on both KCl and methacholine-induced contraction). There was no significant difference in the relaxant effects between C. longa and theophylline in both methacholine and KCl-induced contraction conditions. In tissues incubated with propranolol, diltiazem, L-NAME and glibenclamide on methacholine-induced contraction and in tissues incubated with atropine, chlorpheniramine, indomethacin and papaverine on KCl-induced contraction, the extract also showed significant concentration-dependent relaxant effects (p < 0.001). EC 50 values of C. longa between non-incubated (16.22 ± 0.62) and incubated tissues (atropine: 13.03 ± 0.55, chlorpheniramine: 12.94 ± 0.68, indomethacin: 14.80 ± 0.57 and papaverine: 16.16 ± 1.42) were not significantly different., Conclusions: Tracheal smooth muscle relaxant effects of C. longa, were comparable to those of theophylline, which could be due to the presence of methylxanthines or its possible interaction with non-adrenergic non-cholinergic nervous system.

Purpose: To evaluate whether or not the bladder function can be protected by supporting the detrusor with decorin levels during the fibrotic process., Methods: Forty-two male rabbits were divided into three main groups, partial bladder outlet obstruction (pBOO) group, pBOO + intradetrusor decorin-injected (IDI) group and control group. Both pBOO and pBOO + IDI groups were divided into three subgroups according to the killing schedule. Histopathological, immunohistochemical and pharmacodynamics studies were performed for the evaluation of fibrotic process and tissue characteristics., Results: Histopathological evaluation revealed statistically significant high fibrosis levels for both pBOO and pBOO + IDI groups when compared with control. Strikingly the antifibrotic effect of decorin was significant on 2nd, 4th and 8th week and increased as time passed. Immunohistochemical analysis was revealed high expressions of anti-TGF-β1 and decorin levels in all pBOO + IDI groups. Pharmacodynamical results were also revealed better contraction responses in favor of 2nd, 4th and 8th week groups of pBOO + IDI groups, when compared with pBOO groups. In addition, the contraction responses against the depolarizer agent KCl were increased in the three decorin-administrated groups., Conclusion: Our study demonstrates the antifibrotic effects of decorin on bladder fibrosis. Strikingly, this antifibrotic effect is shown in histopathological, immunohistochemical and pharmacodynamics studies. Although further studies are warranted to make more decisive inferences regarding its clinical use, our study has the proper pride to be the first step of this time course.

Urinary bladder function consists in the storage and controlled voiding of urine. Translational studies require animal models that match human characteristics, such as Octodon degus, a diurnal rodent. This study aims to characterize the contractility of the detrusor muscle and the morphology and code of the vesical plexus from O. degus. Body temperature was measured by an intra-abdominal sensor, the contractility of detrusor strips was evaluated by isometric tension recording, and the vesical plexus was studied by electrical field stimulation (EFS) and immunofluorescence. The animals showed a diurnal chronotype as judged from core temperature. The myogenic contractile response of the detrusor muscle to increasing doses of KCl reached its maximum (31.04 mN/mm 2 ) at 60 mM. In the case of cumulative dose-response of bethanecol, the maximum response (37.42 mN/mm 2 ) was reached at 3.2 × 10 -4  M. The response to ATP was clearly smaller (3.8 mN/mm 2 ). The pharmacological dissection of the EFS-induced contraction identified ACh and sensory fibers as the main contributors to this response. The neurons of the vesical plexus were located mainly in the trigone area, grouped in big and small ganglia. Out of them, 48.1 % of the neurons were nitrergic and 62.7 % cholinergic. Our results show functional and morphological similarities between the urinary bladder of O. degus and that of humans.

Eyes are supplied O 2 through the cornea and vessels of the retina and iris, which are tissues characterized by aerobic metabolism. Meanwhile, there are no reports on the association between iris sphincter contraction and aerobic metabolism. In this paper, we studied the aforementioned association. Eyes from adult pigs of either sex were obtained from a local abattoir. A muscle strip was connected to a transducer to isometrically record the tension. O 2 consumption was measured using a Clark-type polarograph connected to a biological oxygen monitor. Creatine phosphate (PCr) and adenosine triphosphate (ATP) contents were measured in the muscle strips by high-performance liquid chromatography (HPLC). Iris sphincter muscles were measured in resting, contractile or hypoxic phases. Contraction was induced by hyperosmotic 65 mM KCl (H-65K + ) or carbachol (CCh), and hypoxia was induced by aeration with N 2 instead of O 2 or by addition of sodium cyanide (NaCN). H-65K + - and CCh-induced muscle contraction, involved increasing O 2 consumption. Hypoxia and NaCN significantly decreased H-65K + - and CCh-induced muscle contraction and/or O 2 consumption and PCr contents. Our results suggest that the contractile behavior in porcine iris sphincter highly depends on mitogen oxidative metabolism.

Aim To investigate the effect of magnesium sulfate (MgSO4) at different doses on isolated tracheal smooth muscle contraction in rats induced by different mechanisms. Methods Twelve rats' tracheas were placed into organ bath. Consecutively, acetylcholine (10-6,10-5,10-4 M), histamine(10-8,10-5,10-3 M) and KCl (30,60 mM) solutions was administered for contractions. MgSO4 from 10-4 to 10-1 M concentrations were subsequently administered after each constrictive agent and relaxation degrees were recorded. Results In the acetylcholine and KCl groups, dose dependent strong contractions were observed, but not in the histamine group and that group was excluded. Significant relaxation occurred with gradually increasing doses of MgSO4. In the high dose KCl group, a slight increase in contractions after the administration of 10-4 and 10-3 M MgSO4 was recorded. Conclusion We suggest that MgSO4 is effective in relaxing airway smooth muscle contractions caused by different factors; however, it must be considered that low doses of MgSO4 may only lead to a slight increase in contractions., (Copyright© by the Medical Assotiation of Zenica-Doboj Canton.)

Key Points: The extent of myosin regulatory light chain phosphorylation (RLC) necessary for smooth muscle contraction depends on the respective activities of Ca(2+) /calmodulin-dependent myosin light chain kinase and myosin light chain phosphatase (MLCP), which contains a regulatory subunit MYPT1 bound to the phosphatase catalytic subunit and myosin. MYPT1 showed significant constitutive T696 and T853 phosphorylation, which is predicted to inhibit MLCP activity in isolated ileal smooth muscle tissues, with additional phosphorylation upon pharmacological treatment with the muscarinic agonist carbachol. Electrical field stimulation (EFS), which releases ACh from nerves, increased force and RLC phosphorylation but not MYPT1 T696 or T853 phosphorylation. The conditional knockout of MYPT1 or the knockin mutation T853A in mice had no effect on the frequency-maximal force responses to EFS in isolated ileal tissues. Physiological RLC phosphorylation and force development in ileal smooth muscle depend on myosin light chain kinase and MLCP activities without changes in constitutive MYPT1 phosphorylation., Abstract: Smooth muscle contraction initiated by myosin regulatory light chain (RLC) phosphorylation is dependent on the relative activities of Ca(2+) /calmodulin-dependent myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP). We have investigated the physiological role of the MLCP regulatory subunit MYPT1 in ileal smooth muscle in adult mice with (1) smooth muscle-specific deletion of MYPT1; (2) non-phosphorylatable MYPT1 containing a T853A knockin mutation; and (3) measurements of force and protein phosphorylation responses to cholinergic neurostimulation initiated by electric field stimulation. Isolated MYPT1-deficient tissues from MYPT1(SM-/-) mice contracted and relaxed rapidly with moderate differences in sustained responses to KCl and carbachol treatments and washouts, respectively. Similarly, measurements of regulatory proteins responsible for RLC phosphorylation during contractions also revealed moderate changes. There were no differences in contractile or RLC phosphorylation responses to carbachol between tissues from normal mice vs. MYPT1 T853A knockin mice. Quantitatively, there was substantial MYPT1 T696 and T853 phosphorylation in wild-type tissues under resting conditions, predicting a high extent of MLCP phosphatase inhibition. Reduced PP1cδ activity in MYPT1-deficient tissues may be similar to attenuated MLCP activity in wild-type tissues resulting from constitutively phosphorylated MYPT1. Electric field stimulation increased RLC phosphorylation and force development in tissues from wild-type mice without an increase in MYPT1 phosphorylation. Thus, physiological RLC phosphorylation and force development in ileal smooth muscle appear to be dependent on MLCK and MLCP activities without changes in constitutive MYPT1 phosphorylation., (© 2016 The Authors. The Journal of Physiology © 2016 The Physiological Society.)

The aim of this study was to evaluate whether the sub-chronic systemic ethanol exposure has direct effect on cavernosal smooth muscle contractions induced by KCl (depolarizing) and phenylephrine (α1-receptor agonist), and the possible involvement of RhoA/Rho-kinase pathway. Sub-chronic systemic ethanol was applied to mice with inhalation route for 14 days. The blood levels in ethanol-treated mice averaged 121.2 ± 9.1 mg/dl. KCl (80 mM) and phenylephrine (10 nM-100 μM) induced sustained contractions in corpus corporal strips from sham-treated mice. Sub-chronic ethanol treatment reduced the contractions to KCl. However, phenylephrine-induced contractions were not affected by ethanol treatment. Rho-kinase inhibitor fasudil (50 μM) and Y-27632 (50 μM) inhibited contractions to KCl and phenylephrine in sham-treated mice. Ethanol treatment increased the inhibitory effect of Rho-kinase inhibitors on contractions to phenylephrine. The relaxations induced by fasudil (100 μM) and Y-27632 (500 μM) did not change in ethanol treatment group. In ethanol-treated group, the expression of RhoA decreased compared to sham-treated group. Also, ROCK1 expression was reduced by ethanol but not statically significant to sham-treated group; however, the expression of ROCK2 increased in ethanol group. From these findings, it seems that phenylephrine and KCl-induced contractions depends on RhoA/Rho-kinase-mediated Ca(2+) sensitization. Also, these results suggest that the ethanol treatment decreased the expression of RhoA, and the inhibitory effect of ethanol on KCl-induced contractions may be due to, at least in part, the inhibition of a RhoA/Rho-kinase in mouse corpus cavernosum.

Therapy with estrogens is frequently used in menopausal women and as hormonal contraception. Because of its thrombotic effects, long term estrogen administration used in hormonal replacement therapy (HRT) and contraception could represent a health hazard. In this regard, 17β-aminoestrogens such as aminoestrol, butolame and pentolame have shown promising HRT potential, because they have a weak agonist estrogenic action and antithrombotic activity. Additionally, estrogens play a protective role in airway smooth muscle, but the effect of 17β-aminoestrogens on the airway smooth muscle has not been tested yet. In guinea pig tracheal smooth muscle pentolame and butolame induced hyperresponsiveness to histamine (His), carbachol (Cch) and KCl. Interestingly, aminoestrol did not show this effect at the highest concentration studied, it even lowered the contraction induced by Cch. The hyperresponsiveness induced by pentolame to His was abolished by nifedipine. In single tracheal myocytes, KCl induced an increment in the intracellular Ca(2+) concentration [Ca(2+)]i, pentolame also showed an increase in [Ca(2+)]i and the addition of KCl in the plateau of this rise further significantly augmented the [Ca(2+)]i response. Additionally, in patch clamp experiments pentolame increased the L-type Ca(2+) currents. Thus, 17β-aminoestrogens such as pentolame and butolame, but not aminoestrol, activate L-type Ca(2+) channel to induced hyperresponsiveness to Cch, His and KCl in guinea pig tracheal smooth muscle. Due to its lack of effect on airways and to its anticoagulant characteristics, aminoestrol seems to be the best alternative in the HRT among the 17β-aminoestrogens studied., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Ethnopharmacological Relevance: Pittosporum mannii Hook. f. (Pittosporaceae) is a plant widely used in traditional medicine in Cameroon for the treatment of many gastrointestinal disorders including diarrhea. To date, no pharmacological study on the antidiarrheal and the antispasmodic properties of this plant has been reported. The aim of the present study was to evaluate in vitro the relaxant activity of the aqueous extract of stem barks of P. mannii (PMAE) on rat duodenum., Materials and Methods: Different concentrations of PMAE were tested separately (10-80 µg/mL) or cumulatively (5-80 µg/mL) on spontaneous and spasmogen (carbachol, histamine and KCl)-induced contractions of isolated rat duodenum strips., Results: At concentrations ranging from 10 to 80 µg/mL, PMAE significantly decreased the tonus and the amplitude of spontaneous contractions. However, at high concentration (80 µg/mL), the extract elicited a transient relaxation was followed by a slight increase of tonus, while the amplitude remained lower compared to the normal spontaneous activity. The relaxant effect of the extract was not significantly affected in the presence of atropine (0.713 µg/mL) and promethazine (0.5 µg/mL). In addition, PMAE (20, 40, and 80 µg/mL) partially but significantly inhibited in a concentration related manner the contractions induced by carbachol (10(-9)-10(-4)M) and histamine (10(-9)-10(-4)M) on rat duodenum. PMAE (10-80 µg/mL) also significantly induced a concentration-dependent relaxation on KCl (20mM, 50mM, 10(-3)-6.10(-3)M)-induced contraction of rat duodenum., Conclusions: These results show that the aqueous extract of P. mannii stem barks possesses antispasmodic and spasmolytic effects at lower concentrations; therefore, supporting the use of the stem barks of this plant in the folk medicine for the treatment of diarrhea. However, caution should be paid while using higher concentrations that instead might produce spasmogenic effect and might worsen the diarrheal condition. The relaxant effect of PMAE appears to be non-specific of muscarinic or histaminic receptors, but may involve at least in part a mechanism of inhibition of the Ca(2+) influx into the smooth muscle cells through voltage-operated Ca(2+) channels., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Background: Ruta graveolens L. (R. graveolens) is a medicinal plant employed in non-traditional medicines that has various therapeutic properties, including anthelmintic, and vasodilatory actions, among others. We evaluated the trachea-relaxant effects of hydroalcoholic extract of R. graveolens against potassium chloride (KCl)- and carbachol-induced contraction of rat tracheal rings in an isolated organ bath., Results: The results showed that the airway smooth muscle contraction induced by the depolarizing agent (KCl) and cholinergic agonist (carbachol) was markedly reduced by R. graveolens in a concentration-dependent manner, with maximum values of 109 ± 7.9 % and 118 ± 2.6 %, respectively (changes in tension expressed as positive percentages of change in proportion to maximum contraction), at the concentration of 45 μg/mL (half-maximal inhibitory concentration IC50: 35.5 μg/mL and 27.8 μg/mL for KCl- and carbachol-induced contraction, respectively). Additionally, the presence of R. graveolens produced rightward parallel displacement of carbachol dose-response curves and reduced over 35 % of the maximum smooth muscle contraction., Conclusions: The hydroalcoholic extract of R. graveolens exhibited relaxant activity on rat tracheal rings. The results suggest that the trachea-relaxant effect is mediated by a non-competitive antagonistic mechanism. More detailed studies are needed to identify the target of the inhibition, and to determine more precisely the pharmacological mechanisms involved in the observed biological effects.

This study examined the hypothesis that the force generated by myometrial strips from pregnant women is influenced by the smooth muscle content and fibre orientation of the strips and that correcting for these structural variables will provide a more accurate measure of contractility. Myometrial strips (n=72) were contracted by exposure to KCl, oxytocin, U44619 and phenylephrine and maximum responses were recorded. Morphological techniques were used to determine the cross-sectional area of the strips, the area occupied by smooth muscle and the area occupied by smooth muscle longitudinal in the strip. Maximum responses to contractile agents were expressed in terms of these three variables. The mean cross sectional area of strips was 2.01 ± 0.06 mm(2), of which 50% was smooth muscle, and 18% was smooth muscle longitudinal in the strip (n=72). There was much heterogeneity in responses, smooth muscle content and fibre orientation. Correction for morphological variability did not improve the heterogeneity in responses where coefficients of variation among strips from the same donor ranged from 43% to 63% when expressed in relation to longitudinal smooth muscle cross-sectional area. The standard method of preparation of myometrial strips for in vitro recording results in samples that are not structurally uniform. Correcting for the known structural variables does not provide a more accurate measure of maximum contractile responses. Because of the heterogeneity shown here, experiments that are dependent upon accurate estimation of maximum contractile responses require a large number of replicates to reach meaningful conclusions., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Background: Immature motility of the ileum may contribute to life-threatening diseases. Little is known about the normal biomechanics of the neonatal ileum in relation to the protein composition of its contractile machinery., Methods: We analyzed the tissue architecture, the biomechanics in intact and β-escin-permeabilized preparations, and the protein composition in neonatal (P0) and adult murine ileum., Results: Muscle thickness of the P0 ileum was -50% of the adult ileum and passive compliance was higher. Carbachol- and KCl-elicited contractions were tonic rather than phasic as in the adult. Ca(2+) sensitivity was higher and relaxation rate was slower in β-escin-permeabilized P0 compared with adult ileum. The expression level of β-actin relative to α-actin was higher, and those of total actin, myosin, myosin light chain kinase, the catalytic subunit of myosin phosphatase and telokin were lower compared with the adult. The expression level of MYPT1 was similar, but P0 ileum expressed only the M133; the adult ileum also expressed the M130 isoform., Conclusion: The mechanical features and protein composition of the P0 ileum are similar to those of adult tonic smooth muscles. We propose that this is highly adaptive during fetal life allowing the small intestine to act predominantly as a container.

Dehydroeffusol is a naturally occurring phenanthrene isolated from Juncus effusus. In the context of screening new drugs against gastrointestinal spasms, we investigated its effects on isolated rat jejunum in vitro. Dehydroeffusol (30-90 µM) slightly and transiently enhanced contractions in a concentration-dependent manner but significantly inhibited the contractions induced by KCl (100 mM), (±)-Bay-K8644 (5 µM), pilocarpine (90 µM), and histamine (100 µM). These results show that dehydroeffusol may antagonize the spasmogenic activity of various agents, and therefore, could be a promising agent in the treatment of spasms. Its potential spasmolytic mechanism is also discussed., (Georg Thieme Verlag KG Stuttgart · New York.)

Changes in the androgen levels in asthmatic men may be associated with the severity of asthma. Androgens induce a nongenomic relaxation in airway smooth muscle, but the underlying mechanisms remain unclear. The aim of this study was to investigate the potential bronchorelaxing action of testosterone (TES) and its metabolites (5α- and 5β-dihydrotestosterone (DHT). A preventive effect on ovalbumin (OVA)-induced bronchospasm was observed in sensitized guinea pigs for each androgen. Androgens were studied in response to bronchoconstrictors: carbachol (CCh) and KCl in isolated trachea rings with and without epithelium from non-sensitized and sensitized animals as well as on OVA-induced contraction. Androgens concentration-dependently abolished the contraction in response to CCh, KCl, and OVA. There were significant differences in the sensitivity to the relaxation induced by each androgen. 5β-DHT was more potent for relaxing KCl-induced contraction, while TES and 5α-DHT were more potent for CCh- and OVA-induced contraction. No differences were found in preparations with and without epithelium or in the presence of a nitric oxide (NO) synthase inhibitor or an inhibitor of K(+) channels. These data indicate the absence of involvement of the epithelium-, NO- and K(+) channels-dependent pathway in androgen-induced relaxation. However, in dissociated tracheal myocytes loaded with the calcium-binding fluorescent dye Fura -2, physiological concentrations of androgens decreased the KCl-induced [Ca(2+)]i increment. 5β-DHT was the most potent at decreasing KCl-induced [Ca(2+)]i increment and preventing bronchospasm. We suggest that androgen-induced brochorelaxation was mediated via decreased Ca(2+) influx through L-type Ca(2+)channels but additional Ca(2+) entry blockade may be involved. Molecular changes in androgen structure may determine its preferential site of action., (© 2014 Society for Endocrinology.)

Purpose: Bowel dilatation occurs proximal to an obstruction and predisposes to intestinal dysmotility. The present study sought to determine whether or not changes in smooth muscle contractility and the thickness of the proximal, dilated bowel wall can be reversed following relief of the obstruction., Materials and Methods: Three groups of seven male Wistar rats were studied. In 8-week-old animals in a control group and a sham-operated group, a small segment of bowel (designated as R1 for controls and R2 for shams) was resected 5.0 cm from the cecum. In the third (operated) group, a narrow, isoperistaltic intestinal loop was created proximal to an end-to-end anastomosis of the ileum in 4-week-old animals. When these animals were 6 weeks old, the loop was re-anastomosed to the distal small bowel (after resection of the loop's distal portion, referred to as R3). Two weeks later, a small segment of bowel was resected proximal to the anastomosis (R4). We evaluated the thickness of the smooth muscle layers and the in vitro contractile responses of circular smooth muscle ileal strips (R1-R4) to electrical stimulation and pharmacological stimulation (with KCl, acetylcholine (ACh), substance P, N(G)-nitro-l-arginine methyl ester (L-NAME) and histamine)., Results: The amplitudes of contraction in response to electrical and Ach-mediated stimulation were higher for R3 than for R4 (P<0.001), R1 and R2 (both P<0.05). Compared with R1 and R2, the smooth muscle layer was three times as thick in R3 (P<0.001) and 2.5 times as thick in R4 (P<0.01)., Conclusion: Our study provides evidence of the possible recovery of intestinal motility (in response to neurotransmitters involved in gut function) after the relief of an obstruction. If ileal motility can conceivably return to normal values, conservative surgical procedures in pediatric patients should be preferred (in order to leave a sufficient length of bowel and avoid short bowel syndrome)., (© 2013 Elsevier Inc. All rights reserved.)

Airway smooth muscle (ASM) membrane depolarization through KCl opens L-type voltage dependent Ca2+ channels (Ca(v)1.2); its opening was considered the cause of KCl contraction. This substance is used to bypass intracellular second messenger pathways. It is now clear that KCl also activates RhoA/Rho kinase (ROCK) pathway. ROCK isoforms are characterized as ROCK1 and ROCK2. Because ROCK1 seems the most abundant isotype in lung, we studied its participation in KCl stimulated bovine ASM. With methyl-beta-cyclodextrin (MbetaCD) we disrupted caveolae, a membrane compartment considered as the RhoA/ROCK assembly site, and found that KCl contraction was reduced to the same extent (~26%) as Y-27632 (ROCK inhibitor) treated tissues. We confirmed that KCl induces ROCK activation and this effect was annulled by Y-27632 or MbetaCD. In isolated plasmalemma, ROCK1 was localized in non-caveolar membrane fractions in Western blots from control tissues, but it transferred to caveolae in samples from tissues stimulated with KCl. Ca(v)1.2 was found at the non-caveolar membrane fractions in control and MbetaCD treated tissues. In MbetaCD treated tissues stimulated with KCl, contraction was abolished by nifedipine; only the response to Ca(v)1.2 opening remained as the ROCK component disappeared. Our results show that, in ASM, the KCl contraction involves the translocation of ROCK1 from non-caveolar to caveolar regions and that the proper physiological response depends on this translocation.

Caldesmon (CaD), a component of smooth muscle thin filaments, binds actin, tropomyosin, calmodulin, and myosin and inhibits actin-activated ATP hydrolysis by smooth muscle myosin. Internal deletions of the chicken CaD functional domain that spans from amino acids (aa) 718 to 731, which corresponds to aa 512-530 including the adjacent aa sequence in mouse CaD, lead to diminished CaD-induced inhibition of actin-activated ATP hydrolysis by myosin. Transgenic mice with mutations of five aa residues (Lys(523) to Gln, Val(524) to Leu, Ser(526) to Thr, Pro(527) to Cys, and Lys(529) to Ser), which encompass the ATPase inhibitory determinants located in exon 12, were generated by homologous recombination. Homozygous (-/-) animals did not develop, but heterozygous (+/-) mice carrying the expected mutations in the CaD ATPase inhibitory domain (CaD mutant) matured and reproduced normally. The peak force produced in response to KCl and electrical field stimulation by the detrusor smooth muscle from the CaD mutant was high compared with that of the wild type. CaD mutant mice revealed nonvoiding contractions during bladder filling on awake cystometry, suggesting that the CaD ATPase inhibitory domain suppresses force generation during the filling phase and this suppression is partially released by mutations in 50% of CaD in heterozygous. Our data show for the first time a functional phenotype, at the intact smooth muscle tissue and in vivo organ levels, following mutation of a functional domain at the COOH-terminal region of CaD.

Achillea millefolium L. is cultivated in Iran and widely used in traditional medicine for gastrointestinal disorders. The aim of this study was to determine the effect of hydroalcoholic extract of A. millefolium on the contraction and relaxation of isolated ileum in rat. In this experimental study, aerial parts of A. millefolium were extracted by maceration in ethanol 70% for 72 h. Terminal portion of ileum in 100 male Wistar rats was dissected and its contractions were recorded isotonically in an organ bath containing Tyrode solution (37 °C, pH 7.4) under one gram tension. Acetylcholine (1mM) and KCl (60mM) were used to create isotonic contractions. Propranolol and Nω-Nitro-L-arginine methylester hydrochloride (L-NAME) were used to investigate the mechanisms of action prior to giving the extract to the relevant groups. Data were compared by ANOVA and Turkey's post hoc test.. The results showed that the ileum contraction was induced by KCl and acetylcholine induced contraction was significantly reduced by A. millefolium extract. The cumulative concentrations of A. millefolium relaxed the KCl and acetylcholine induced contractions (n=14, p<0.001). The inhibitory effect of extract on contraction induced by KCl and acetylcholine was not significantly affected neither by propranolol (1µM) nor by L-NAME (100 µM). There was no significant difference in the rate of relaxation by propranolol and L-NAME between the two groups. In conclusion, A. millefolium can inhibit contraction of smooth muscle of ileum in rat, and it can be used for eliminating intestinal spasms. These results suggest that the relaxatory effect of A. millefolium on ileum contractions can be due to the blockade of voltage dependent calcium channels. In addition, the β-adrenoceptors, cholinergic receptors and nitric oxide production are not powerful actors in inhibitory effect of A. millefolium. So, the nitric oxide and adrenergic systems may also be involved in the antispasmodic effect of A. millefolium.

Kv7 channels are involved in smooth muscle relaxation, and accordingly we believe that they constitute potential targets for the treatment of overactive bladder syndrome. We have therefore used myography to examine the function of Kv7 channels in detrusor, i.e. pig bladder, with a view to determining the effects of the following potassium channel activators: ML213 (Kv7.2/Kv7.4 channels) and retigabine (Kv7.2-7.5 channels). Retigabine produced a concentration-dependent relaxation of carbachol- and electric field-induced contractions. The potency was similar in magnitude to that of ML213-induced relaxation, suggesting that Kv7.2 and/or Kv7.4 channels constitute the subtypes that are relevant to bladder contractility. The effects of retigabine and ML213 were attenuated by pre-incubation with 10µM XE991 (Kv7.1-7.5 channel blocker) (P<0.05), which in turn confirmed Kv7 channel selectivity. Subtype-selective effects were further investigated by incubating the detrusor with 10µM chromanol 293B (Kv7.1 channel blocker). Regardless of the experimental protocol, this did not cause a further increase in the evoked contraction. In contrast, the addition of XE991 potentiated the KCl-induced contractions, but not those induced by carbachol or electric field, indicating the presence of a phosphatidyl-inositol-4,5-biphosphate-dependent mechanism amongst the Kv7 channels in detrusor. qRT-PCR studies of the mRNA transcript level of Kv7.3-7.5 channels displayed a higher level of Kv7.4 transcript in detrusor compared to that present in brain cortex and heart tissues. Thus, we have shown that Kv7.4 channels are expressed and functionally active in pig detrusor, and that the use of selective Kv7.4 channel modulators in the treatment of detrusor overactivity seems promising., (© 2013 Elsevier B.V. All rights reserved.)

Background/purpose: The 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., Methods: Sham (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., Results: SC 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., Conclusions: UVJ 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., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The objective of this study was to define the regional differences in rat vaginal smooth muscle contractility and morphology. We evaluated circumferential segments from the proximal, middle, and distal rat vagina (n = 21) in vitro. Contractile responses to carbachol, phenylephrine, potassium chloride, and electrical field stimulation (EFS) were measured. Immunohistochemical analyses were also performed. The dose-response curves for carbachol- and phenylephrine-dependent contractions were different in the distal (P = .05, P = .04) compared to the proximal/middle regions. Adjusted for region-dependent changes in contractility, the distal vagina generated lower force in response to carbachol and higher force in response to phenylephrine. There was less force with increasing EFS frequency in the distal (P = .03), compared to the proximal/middle regions. Cholinergic versus adrenergic nerves were more frequent in the proximal region (P = .03). In summary, the results indicate that functional and morphological differences in smooth muscle and nerve fibers of the distal versus proximal/middle regions of the vagina exist.

In rabbit bladder wall (detrusor) muscle, the degree of tone induced during physiological filling (filling tone) is the sum of adjustable preload tension and autonomous contractile tension. The present study was designed to determine whether the level of filling tone is dependent on detrusor muscle length. Maximum active tension induced by KCl was parabolic in relation to length [tension increased from 70% to 100% of a reference length (L(ref)) and decreased at longer muscle lengths]. Filling tone, however, increased in a linear fashion from 70% to 120% L(ref). In the presence of ibuprofen to abolish autonomous contraction and retain adjustable preload tension, tension was reduced in strength but remained linearly dependent on length from 70% to 120% L(ref). In the absence of autonomous contraction, stretching detrusor muscle from 80% to 120% L(ref) still caused an increase in tone during PGE(2)-induced rhythmic contraction, suggesting that muscle stretch caused increases in detrusor muscle contractile sensitivity rather than in prostaglandin release. In the absence of autonomous contraction, the degree of adjustable preload tension and myosin phosphorylation increased when detrusor was stretched from 80% to 120% L(ref), but also displayed length-hysteresis, indicating that detrusor muscle senses preload rather than muscle length. Together, these data support the hypothesis that detrusor muscle acts as a preload tension sensor. Because detrusor muscle is in-series with neuronal mechanosensors responsible for urinary urgency, a more thorough understanding of detrusor muscle filling tone may reveal unique targets for therapeutic intervention of contractile disorders such as overactive bladder.

Aim: An in vitro study was performed to determine the in vitro sensitivity of rat esophagi to contracting and relaxing agonists in different alkaline mediums., Materials and Methods: Twenty-four Wistar rats weighing 200 to 250 g were included in the study. After inducing anesthesia with thiopental sodium (50 mg/kg), the rats were killed, and the distal esophagus (DE) and gastroesophageal junction (GEJ) were removed. Excised tissues were suspended under 0.6 g of resting tension in a tissue bath containing 10 mL of Tyrode solution at 37°C. The esophageal segments were divided into 4 groups, each with a different pH medium: 7.4, 7.6, 7.8, and 8. To evaluate the smooth muscle activity of the rat esophagus in different alkaline mediums, cholinergic agonist carbachol (a cholinergic agonosit) and KCl (which acts on calcium channels) were added to the organ baths to obtain concentration-dependent contraction responses. Relaxation responses were obtained using an adrenergic agonist, isoproterenole. Data obtained from different pH mediums were analyzed using Kruskal-Wallis nonparametric analysis of variance and post hoc Dunn's test., Results: The pH of the medium has no effect on the concentration-dependent contracting responses to carbachol in the DE, but responses obtained in pH 8 medium were significantly lower in the GEJ (P < .05). In pH 8 medium, concentration-dependent relaxation responses to isoproterenole were also significantly lower (P < .05). When contractile responses to KCl were evaluated, the groups with pH 7.6 and 7.8 medium were found to have higher responses in the DE segments than other groups (P < .05). However, the GEJ segments had lower contractile responses to KCl in pH 8 medium (P < .05)., Conclusion: Although different alkaline mediums caused no alteration in esophageal smooth muscle responses to cholinergic stimulation, GEJ responses were lower in pH 8 medium. Relaxation responses, owing to increased adrenergic activity, decreased when the pH of the medium was increased to 8. Calcium-dependent contractions caused by KCl decreased in both segments in pH 8 medium but increased in the DE in pH 7.6 medium., (Copyright © 2012 Elsevier Inc. All rights reserved.)

To elucidate the role of glycogen in the contraction of tracheal smooth muscle, we investigated the changes in the glycogen contents of the bovine trachea during contractions induced by high K(+) and hypoxia (achieved by bubbling N(2) instead of O(2)), either in a glucose-free condition or in the presence of iodoacetic acid (IAA), an inhibitor of glycolysis. Hyperosmotic addition of 65 mM KCl (H-65 K(+)) induced a sustained contraction. A glucose-free condition did not affect H-65 K(+)-induced contraction. However, hypoxia slightly inhibited the contraction, and glucose-free PSS with hypoxia or IAA remarkably inhibited the H-65 K(+)-induced contraction. H-65 K(+) induced a sustained increase in reduced pyridine nucleotide (PNred) fluorescence, representing glycolysis activity. Hypoxia alone slightly enhanced PNred fluorescence, and when combined with a glucose-free condition, it remarkably enhanced the H-65 K(+)-induced PNred fluorescence. IAA inhibited PNred fluorescence. In the presence of H-65 K(+), a glucose-free condition, hypoxia and the combination of glucose-free PSS and hypoxia decreased the glycogen contents. However, IAA had no effect on glycogen contents. Although hypoxia or glucose-free PSS did not affect PCr and ATP contents, the combination of hypoxia and glucose-free PSS or IAA induced a gradual decrease of PCr content. In conclusion, we suggest that endogenous glycogen was utilized to increase the activity of glycolysis for maintaining high K(+)-induced contraction of the bovine trachea in the glucose -free and/or hypoxic condition.

The antispasmodic effect of 4'-methylepigallocatechin (MEC), which was isolated from Maytenus rigida Mart (Celestraceae), was investigated in vitro in guinea pig intestinal segments. In the isolated ileum, MEC (1 nM-100 μM) did not modify the ileal spontaneous tonus or the electrically elicited contractions. MEC (8 μM) significantly (p<0.01) reduced the submaximal contractions induced by histamine (2 μM), carbachol (100 μM) and BaCl₂ (0.03 M). An additive relaxing action (p<0.001) was observed by co-incubation of verapamil (10 nM) and MEC (8 μM). Although MEC (1 nM-100 μM) did not modify the contractions elicited by 60 mM KCl, it significantly reduced the CaCl₂ contractile response without changing the EC₅₀ (effective concentration of CaCl₂ causing 50% of maximum response). In brief, these results show that MEC has a potent ileal spasmolytic effect and blocks spasms induced by specific and nonspecific stimuli. Importantly, the spasmolytic effects were attained at low concentrations and might be related to the symptomatic relief of abdominal pain that is obtained from the use of the M. rigida stem bark., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Caffeine is sometimes used in cell physiological studies to release internally stored Ca(2+). We obtained evidence that caffeine may also act through a different mechanism that has not been previously described and sought to examine this in greater detail. We ruled out a role for phosphodiesterase (PDE) inhibition, since the effect was 1) not reversed by inhibiting PKA or adenylate cyclase; 2) not exacerbated by inhibiting PDE4; and 3) not mimicked by submillimolar caffeine nor theophylline, both of which are sufficient to inhibit PDE. Although caffeine is an agonist of bitter taste receptors, which in turn mediate bronchodilation, its relaxant effect was not mimicked by quinine. After permeabilizing the membrane using β-escin and depleting the internal Ca(2+) store using A23187, we found that 10 mM caffeine reversed tone evoked by direct application of Ca(2+), suggesting it functionally antagonizes the contractile apparatus. Using a variety of molecular techniques, we found that caffeine did not affect phosphorylation of myosin light chain (MLC) by MLC kinase, actin-filament motility catalyzed by MLC kinase, phosphorylation of CPI-17 by either protein kinase C or RhoA kinase, nor the activity of MLC-phosphatase. However, we did obtain evidence that caffeine decreased actin filament binding to phosphorylated myosin heads and increased the ratio of globular to filamentous actin in precontracted tissues. We conclude that, in addition to its other non-RyR targets, caffeine also interferes with actin function (decreased binding by myosin, possibly with depolymerization), an effect that should be borne in mind in studies using caffeine to probe excitation-contraction coupling in smooth muscle.

Detrusor smooth muscle (DSM) contributes to bladder wall tension during filling, and bladder wall deformation affects the signaling system that leads to urgency. The length-passive tension (L-T(p)) relationship in rabbit DSM can adapt with length changes over time and exhibits adjustable passive stiffness (APS) characterized by a L-T(p) curve that is a function of both activation and strain history. Muscle activation with KCl, carbachol (CCh), or prostaglandin E(2) at short muscle lengths can increase APS that is revealed by elevated pseudo-steady-state T(p) at longer lengths compared with prior T(p) measurements at those lengths, and APS generation is inhibited by the Rho Kinase (ROCK) inhibitor H-1152. In the current study, mouse bladder strips exhibited both KCl- and CCh-induced APS. Whole mouse bladders demonstrated APS which was measured as an increase in pressure during passive filling in calcium-free solution following CCh precontraction compared with pressure during filling without precontraction. In addition, CCh-induced APS in whole mouse bladder was inhibited by H-1152, indicating that ROCK activity may regulate bladder compliance during filling. Furthermore, APS in whole mouse bladder was elevated 2 wk after partial bladder outlet obstruction, suggesting that APS may be relevant in diseases affecting bladder mechanics. The presence of APS in mouse bladder will permit future studies of APS regulatory pathways and potential alterations of APS in disease models using knockout transgenetic mice.

Hypotension is a frequent side effect of the antidepressant treatment. It is controversial whether this effect is attributable to interactions within the central nervous or the cardiovascular system. We examined often used antidepressants for their vasoactive properties in vitro in rat aortal rings with and without endothelium. The influence of pre-incubation with the antidepressants (0.5 μM) on adrenergic elicited smooth muscle contraction and the effects of cumulative concentrations (0.05 μM-500 μM) of the antidepressants on isometric tension were measured. In addition, conceivable modulation of the NO-cGMP, adrenergic and potassium channel pathways were examined. Amitriptyline and fluoxetine inhibited, whereas tranylcypromine enhanced adrenergic elicited responses of smooth muscle contraction. The antidepressants amitriptyline, fluoxetine and tranylcypromine showed, to a different extent, vasorelaxing properties in the preparations pre-contracted with phenylephrine 0.1 μM; the pEC50, (means and S.E.M.) in descending order of potency: amitriptyline 6.98 (0.13), fluoxetine 6.11 (0.05), tranylcypromine 5.33 (0.05) (n=8 each, preparations with endothelium); or after pre-contraction with KCl 20 mM: fluoxetine 6.00 (0.06), tranylcypromine 4.99 (0.30), amitriptyline, 4.89 (0.11), (n=7 each, preparations with endothelium). Venlafaxine did not relax the aortal rings and even lead to further contraction of the endothelium intact preparations. The observed effects were partially endothelium dependent via activation of the NO-cGMP pathway and some probably mediated through K+ channel activation. Amitriptyline, fluoxetine and tranylcypromine relax rat aorta in vitro. They partially delay vascular smooth muscle reactions to adrenergic agonists and can lead to sustained hypotension episodes despite administration of sympathomimetic drugs.

Emerging evidence suggests that the T helper 17 (T(H)17) subset of αβ T cells contributes to the development of allergic asthma. In this study, we found that mice lacking the αvβ8 integrin on dendritic cells did not generate T(H)17 cells in the lung and were protected from airway hyper-responsiveness in response to house dust mite and ovalbumin sensitization and challenge. Because loss of T(H)17 cells inhibited airway narrowing without any obvious effects on airway inflammation or epithelial morphology, we examined the direct effects of T(H)17 cytokines on mouse and human airway smooth muscle function. Interleukin-17A (IL-17A), but not IL-17F or IL-22, enhanced contractile force generation of airway smooth muscle through an IL-17 receptor A (IL-17RA)-IL-17RC, nuclear factor κ light-chain enhancer of activated B cells (NF-κB)-ras homolog gene family, member A (RhoA)-Rho-associated coiled-coil containing protein kinase 2 (ROCK2) signaling cascade. Mice lacking integrin αvβ8 on dendritic cells showed impaired activation of this pathway after ovalbumin sensitization and challenge, and the diminished contraction of the tracheal rings in these mice was reversed by IL-17A. These data indicate that the IL-17A produced by T(H)17 cells contributes to allergen-induced airway hyper-responsiveness through direct effects on airway smooth muscle.

Voltage-gated K(+) (K(V)) channels are implicated in detrusor smooth muscle (DSM) function. However, little is known about the functional role of the heterotetrameric K(V) channels in DSM. In this report, we provide molecular, electrophysiological, and functional evidence for the presence of K(V)2.1 and electrically silent K(V) channel subunits in guinea pig DSM. Stromatoxin-1 (ScTx1), a selective inhibitor of the homotetrameric K(V)2.1, K(V)2.2, and K(V)4.2 as well as the heterotetrameric K(V)2.1/6.3 and K(V)2.1/9.3 channels, was used to examine the role of these K(V) channels in DSM function. RT-PCR indicated mRNA expression of K(V)2.1, K(V)6.2-6.3, K(V)8.2, and K(V)9.1-9.3 subunits in isolated DSM cells. K(V)2.1 protein expression was confirmed by Western blot and immunocytochemistry. Perforated whole cell patch-clamp experiments revealed that ScTx1 (100 nM) inhibited the amplitude of the K(V) current in freshly isolated DSM cells. ScTx1 (100 nM) did not significantly change the steady-state activation and inactivation curves for K(V) current. However, ScTx1 (100 nM) decreased the activation time-constant of the K(V) current at positive voltages. Although our patch-clamp data could not exclude the presence of the homotetrameric K(V)2.1 channels, the biophysical characteristics of the ScTx1-sensitive current were consistent with the presence of heterotetrameric K(V)2.1/silent K(V) channels. Current-clamp recordings showed that ScTx1 (100 nM) did not change the DSM cell resting membrane potential. ScTx1 (100 nM) increased the spontaneous phasic contraction amplitude, muscle force, and muscle tone as well as the amplitude of the electrical field stimulation-induced contractions of isolated DSM strips. Collectively, our data revealed that K(V)2.1-containing channels are important physiological regulators of guinea pig DSM excitability and contractility.

Synthetic ion channels may have potential therapeutic applications, provided they possess appropriate biological activities. The present study was designed to examine the ability of small molecule-based synthetic Cl(-) channels to modulate airway smooth muscle responsiveness. Changes in isometric tension were measured in rat tracheal rings. Relaxations to the synthetic chloride channel SCC-1 were obtained during sustained contractions to KCl. The anion dependency of the effect of SCC-1 was evaluated by ion substitution experiments. The sensitivity to conventional Cl(-) transport inhibitors was also tested. SCC-1 caused concentration-dependent relaxations during sustained contractions to potassium chloride. This relaxing effect was dependent on the presence of extracellular Cl(-) and HCO(3) (-). It was insensitive to conventional Cl(-) channels/transport inhibitors that blocked the cystic fibrosis transmembrane conductance regulator and calcium-activated Cl(-) channels. SCC-1 did not inhibit contractions induced by carbachol, endothelin-1, 5-hydroxytryptamine or the calcium ionophore A23187. SCC-1 relaxes airway smooth muscle during contractions evoked by depolarizing solutions. The Cl(-) conductance conferred by this synthetic compound is distinct from the endogenous transport systems for chloride anions.

Estrogen has been shown to have an inhibitory effect on the contractility of gastrointestinal smooth muscle, including the gallbladder. Since estrogen and progesterone levels are elevated during pregnancy, a biliary stasis may develop during pregnancy that is characterized by an increase in the fasting and residual volumes and by a decrease in emptying capacity. This study investigates the effect of 17β-estradiol (E2) on contraction in male guinea pig gallbladder strips. E2 induced a concentration-dependent relaxation of either CCK-induced tension or KCl-induced tension. Pretreatment of the strips with PKA inhibitor 14-22 amide myristolated had no significant effect on the E2-induced relaxation. Pretreatment of strips with 2-APB, and inhibitor of IP(3) induced Ca(2+) release, produced a significant (p<0.001) increase in the amount of E2-induced relaxation when either CCK or KCl were used to induce tension. KT5823, an inhibitor of PKG, also significantly (p<0.001) increased the amount of E2-induced relaxation. Genistein, an inhibitor of protein tyrosine kinase, had no significant effect on the E2-induced relaxation. Bisindolymaleimide IV and chelerythrine Cl- when used in combination had no significant effect on the amount of CCK-induced tension, but significantly (p<0.001) increased the amount of E2-induced relaxation. When E2 was added to the chambers prior to either CCK or KCl, a significant decrease (p<0.001) in the amount of tension generated was observed. The inhibition of extracellular Ca(2+) entry mediates the E2-induced relaxation of CCK- and KCl-induced tension in male guinea pig gallbladder strips., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The aim of this study was to compare the contractility of the anterior vaginal muscularis (AVM) from women with and without pelvic organ prolapse (POP). In vitro experiments were performed to measure the peak force generated in response to potassium chloride (KCl; 125 mmol/L) and phenylephrine by AVM tissue from women with and without POP. Cross-sectional areas and co-localization of α(1A) adrenergic receptor protein with smooth muscle α-actin in AVM strips were determined by histology and immunofluorescence, respectively. There were no differences in the mean amplitude of force generated in response to KCl normalized to either wet weight or muscle cross-sectional area (mN/mm(2)) between women with and without POP (P > .30). However, AVM from women with prolapse produced a significantly higher mean force to KCl normalized to total cross-sectional area compared to controls (P = .007). While the control samples demonstrated a consistent response to phenylephrine, there was no response to this stimulant generated by AVM tissue from women with POP. The proportion of co-localized α(1A) adrenergic receptors with smooth muscle α actin in AVM tissue was significantly less in women with POP compared to normal controls (P < .0001). Although there was significantly greater tissue stress generated by AVM from women with prolapse compared to controls, there were no differences in muscle stress. Absent response to phenylephrine by AVM from women with prolapse may be related to a lower expression of α(1A) adrenergic receptors in vaginal smooth muscle.

Non-specific TRPM8 agonist menthol was shown to inhibit voltage- and agonist-evoked contractions of the smooth muscle (SM) of rat vas deferens. Here we compared the action of menthol with the action of more specific TRPM8 agonist icilin on depolarization- (60 mM KCl), carbachol-(CCh) and noradrenalin-(Nor)-evoked contractions of the SM strips from the prostatic and epididymal portions of the vas deferens of normal and castrated (60-137 days) rats. Inhibitory action of menthol (100 microM) and icilin (10 microM) on the amplitude of KCl-, CCh- and Nor-induced contractions of normal as well as castrated rats was similar consisting about 50%, despite castration per se strongly potentiated CCh- and Nor-evoked contractions compared to the control animals. In the epididymal portion of the control animals menthol suppressed KC 1- and CCh-evoked contractions by 46 +/- 5% and 32 +/- 3% and icilin by only 14 +/- 4% and 6 +/- 7%, respectively, whilst after castration both compounds became virtually ineffective. Considering that TRPM8 may localize in the sarcolemma and sarcoplasmic reticulum (SR) membrane and that menthol can also block voltage-gated calcium channels (VGCCs), our data indicate that in the prostatic portion TRPM8 modulates contractility by primarily decreasing the SR Ca2+ stores content, whilst in the epididymal one by both decreasing the SR filling and supporting Ca2+ entry. Drop in the circulation androgens as a result of castration changes the menthol- and icilin-mediated modulation of the rat vas deferens SM contractility via the decrease of the expression of L-type VGCCs and increase of the expression of TRPM8.

Contraction of detrusor smooth muscle (DSM) at short muscle lengths generates a stiffness component we termed adjustable passive stiffness (APS) that is retained in tissues incubated in a Ca(2+)-free solution, shifts the DSM length-passive tension curve up and to the left, and is softened by muscle strain and release (strain softened). In the present study, we tested the hypothesis that APS is due to slowly cycling actomyosin cross bridges. APS and active tension produced by the stimulus, KCl, displayed similar length dependencies with identical optimum length values. The myosin II inhibitor blebbistatin relaxed active tension maintained during a KCl-induced contraction and the passive tension maintained during stress-relaxation induced by muscle stretch in a Ca(2+)-free solution. Passive tension was attributed to tension maintaining rather than tension developing cross bridges because tension did not recover after a rapid 10% stretch and release as it did during a KCl-induced contraction. APS generated by a KCl-induced contraction in intact tissues was preserved in tissues permeabilized with Triton X-100. Blebbistatin and the actin polymerization inhibitor latrunculin-B reduced the degree of APS generated by a KCl-induced contraction. The degree of APS generated by KCl was inhibited to a greater degree than was the peak KCl-induced tension by rhoA kinase and cyclooxygenase inhibitors. These data support the hypothesis that APS is due to slowly cycling actomyosin cross bridges and suggest that cross bridges may play a novel role in DSM that uniquely serves to ensure proper contractile function over an extreme working length range.

The effects of melatonin and quercetin on the contractile responses of cisplatin-treated rat detrusor smooth muscle were tested. Detrusor strips obtained from four separate rat groups (control, cisplatin, melatonin+cisplatin and quercetin+cisplatin) were mounted in 25 mL organ baths containing Krebs-Henseleit solution (KHS) at 37°C, continuously gassed with 95% O₂ and 5% CO₂. The vasoconstriction induced by acetylcholine (ACh) and potassium chloride (KCl) were compared within the groups. Furthermore, histopathological parameters such as edema, congestion, inflammatory cells, microvascular proliferation, fibrosis, eosinophil, mast cells and epithelial damage were noted. In routine experiments ACh and KCl triggered concentration-dependent contractions. Pretreatment with cisplatin increased the sensitivity but not the maximal response to ACh and KCl. In rats treated with melatonin or quercetin before cisplatin, the EC₅₀ values, but not the maximal response, to both agents were significantly higher than in the cisplatin-treated (CII) group. Histopathological parameters such as edema, congestion, inflammatory cells, microvascular proliferation, fibrosis, eosinophil, mast cells and epithelial damage were all higher in the cisplatin-treated group than in the controls. Melatonin pretreatment significantly decreased mast cell numbers and epithelial damage when compared to cisplatin treatment alone but these effects were not recorded with quercetin pretreatment. These results demonstrate for the first time that melatonin can attenuate urinary bladder injury produced by cisplatin treatment., (Copyright 2010 Prous Science, S.A.U. or its licensors. All rights reserved.)

We examined the contribution of p70 ribosomal S6 kinase (p70S6K) to airway smooth muscle hypertrophy, a structural change found in asthma. In human airway smooth muscle cells, transforming growth factor (TGF)-beta, endothelin-1, and cardiotrophin-1 each induced phosphorylation of p70S6K and ribosomal protein S6 while increasing cell size, total protein synthesis, and relative protein abundance of alpha-smooth muscle actin and SM22. Transfection of myocytes with siRNA against either p70S6K or S6, or infection with retrovirus encoding a kinase-dead p70S6K, reduced cell size and protein synthesis but had no effect on contractile protein expression per mg total protein. Infection with a retrovirus encoding a constitutively active, rapamycin-resistant (RR) p70S6K increased cell size but not contractile protein expression. siRNA against S6 decreased cell size in myocytes expressing RR p70S6K. Finally, TGF-beta treatment, but not RR p70S6K expression, increased KCl-induced fractional shortening. Together, these data suggest that p70S6K activation is both required and sufficient for airway smooth muscle cell size enlargement but not contractile protein expression. Further, ribosomal protein S6 is required for p70S6K-mediated cell enlargement. Finally, we have shown for the first time in a functional cell system that p70S6K-mediated myocyte enlargement alone, without preferential contractile protein expression, is insufficient for increased cell shortening.

Background: A major feature of chronic obstructive pulmonary disease (COPD) is airway remodelling, which includes an increased airway smooth muscle (ASM) mass. The mechanisms underlying ASM remodelling in COPD are currently unknown. We hypothesized that cigarette smoke (CS) and/or lipopolysaccharide (LPS), a major constituent of CS, organic dust and gram-negative bacteria, that may be involved in recurrent airway infections and exacerbations in COPD patients, would induce phenotype changes of ASM., Methods: To this aim, using cultured bovine tracheal smooth muscle (BTSM) cells and tissue, we investigated the direct effects of CS extract (CSE) and LPS on ASM proliferation and contractility., Results: Both CSE and LPS induced a profound and concentration-dependent increase in DNA synthesis in BTSM cells. CSE and LPS also induced a significant increase in BTSM cell number, which was associated with increased cyclin D1 expression and dependent on activation of ERK 1/2 and p38 MAP kinase. Consistent with a shift to a more proliferative phenotype, prolonged treatment of BTSM strips with CSE or LPS significantly decreased maximal methacholine- and KCl-induced contraction., Conclusions: Direct exposure of ASM to CSE or LPS causes the induction of a proliferative, hypocontractile ASM phenotype, which may be involved in airway remodelling in COPD.

The present study was conducted to investigate the reactivity of the corpus cavernosum smooth muscle after unilateral cavernous nerve neurotomy in rabbits. Rabbits (18) were randomly divided into two groups: sham-operated (n = 9) and those subjected to unilateral neurotomy of a 5-mm segment of the cavernous nerve (n = 9). The reactivity of the corpus cavernosum tissue from the neurotomized and sham groups was studied in organ chambers at 4 weeks postoperation. In the neurotomized group, endothelium-dependent relaxation of the corpus cavernosum smooth muscle to carbachol was significantly increased when compared to the sham group. In addition, the sensitivity (i.e., pD(2)) of neurotomized strips to carbachol was also increased when compared to controls. Electrical field stimulation-induced neurogenic relaxation was significantly reduced in the neurotomized group. Relaxation to the nitric oxide (NO) donor sodium nitroprusside and to papaverine was similar in the cavernosal tissue of both groups. There was no change in agonist potency. Furthermore, neurotomy had no effect on KCl-induced contractile responses. When tissue contraction was induced with phenylephrine to study relaxation to various stimuli, the tension induced was similar in the neurotomized and the sham control groups. We conclude that unilateral, chronic cavernous nerve neurotomy causes significant functional changes to the penile erectile tissue of rabbits, which may contribute to the development of impotence., (Copyright 2010 Prous Science, S.A.U. or its licensors. All rights reserved.)

The length-tension (L-T) relationships in airway and vascular smooth muscles have been shown to adapt with length changes over time. Our prior studies have shown that the active and passive L-T relationships in rabbit detrusor smooth muscle (DSM) can adapt and that DSM exhibits adjustable passive stiffness (APS) characterized by a passive L-T curve that is a function of strain and activation history. The present study demonstrates that passive tension due to APS can represent a substantial fraction of total tension over a broad length range. Our previous studies have shown that maximal KCl-induced contractions at short muscle lengths generate APS that is revealed by increased pseudo-steady-state passive tension at longer lengths compared with previous measurements at those lengths. The objective of the present study was to determine the mechanisms involved in APS generation. Increasing the number of KCl-induced contractions or the duration of a contraction increased the amount of APS generated. Furthermore, a fraction of APS was restored in calcium-free solution and was sensitive to the general serine and threonine protein kinase inhibitor staurosporine. Most importantly, rhythmic contraction (RC) generated APS, and because RC occurs spontaneously in human bladder, a physiological role for RC was potentially identified.