Basal Tension Research Articles

A Microfabricated Platform to Measure and Manipulate the Mechanics of Engineered Cardiac Microtissues

Engineered myocardial tissues can be used to elucidate fundamental features of myocardial biology, develop organotypic in vitro model systems, and as engineered tissue constructs for replacing damaged heart tissue in vivo. However, a key limitation is an inability to test the wide range of parameters (cell source, mechanical, soluble and electrical stimuli) that might impact the engineered tissue in a high-throughput manner and in an environment that mimics native heart tissue. Here we used microelectromechanical systems technology to generate arrays of cardiac microtissues (CMTs) embedded within three-dimensional micropatterned matrices. Microcantilevers simultaneously constrain CMT contraction and report forces generated by the CMTs in real time. We demonstrate the ability to routinely produce ~200 CMTs per million cardiac cells (<1 neonatal rat heart) whose spontaneous contraction frequency, duration, and forces can be tracked. Independently varying the mechanical stiffness of the cantilevers and collagen matrix revealed that both the dynamic force of cardiac contraction as well as the basal static tension within the CMT increased with boundary or matrix rigidity. Cell alignment is, however, reduced within a stiff collagen matrix; therefore, despite producing higher force, CMTs constructed from higher density collagen have a lower cross-sectional stress than those constructed from lower density collagen. We also study the effect of electrical stimulation on cell alignment and force generation within CMTs and we show that the combination of electrical stimulation and auxotonic load strongly improves both the structure and the function of the CMTs. Finally, we demonstrate the suitability of our technique for high-throughput monitoring of drug-induced changes in spontaneous frequency or contractility in CMTs as well as high-speed imaging of calcium dynamics using fluorescent dyes. Together, these results highlight the potential for this approach to quantitatively demonstrate the impact of physical parameters on the maturation, structure, and function of cardiac tissue and open the possibility to use high-throughput, low volume screening for studies on engineered myocardium.

Contribution of transient and sustained calcium influx, and sensitization to depolarization-induced contractions of the intact mouse aorta

BackgroundElectrophysiological studies of L-type Ca2+ channels in isolated vascular smooth muscle cells revealed that depolarization of these cells evoked a transient and a time-independent Ca2+ current. The sustained, non-inactivating current occurred at voltages where voltage-dependent activation and inactivation overlapped (voltage window) and its contribution to basal tone or active tension in larger multicellular blood vessel preparations is unknown at present. This study investigated whether window Ca2+ influx affects isometric contraction of multicellular C57Bl6 mouse aortic segments.ResultsIntracellular Ca2+ (Cai2+, Fura-2), membrane potential and isometric force were measured in aortic segments, which were clamped at fixed membrane potentials by increasing extracellular K+ concentrations. K+ above 20 mM evoked biphasic contractions, which were not affected by inhibition of IP3- or Ca2+ induced Ca2+ release with 2-aminoethoxydiphenyl borate or ryanodine, respectively, ruling out the contribution of intracellular Ca2+ release. The fast force component paralleled Cai2+ increase, but the slow contraction coincided with Cai2+ decrease. In the absence of extracellular Ca2+, basal tension and Cai2+ declined, and depolarization failed to evoke Cai2+ signals or contraction. Subsequent re-introduction of external Ca2+ elicited only slow contractions, which were now matched by Cai2+ increase. After Cai2+ attained steady-state, isometric force kept increasing due to Ca2+- sensitization of the contractile elements. The slow force responses displayed a bell-shaped voltage-dependence, were suppressed by hyperpolarization with levcromakalim, and enhanced by an agonist of L-type Ca2+ channels (BAY K8644).ConclusionThe isometric response of mouse aortic segments to depolarization consists of a fast, transient contraction paralleled by a transient Ca2+ influx via Ca2+ channels which completely inactivate. Ca2+ channels, which did not completely inactivate during the depolarization, initiated a second, sustained phase of contraction, which was matched by a sustained non-inactivating window Ca2+ influx. Together with sensitization, this window L-type Ca2+ influx is a major determinant of basal and active tension of mouse aortic smooth muscle.

Effect of 100&amp;deg;C Heating on Further Ductility Improvement of Friction Stir Processed AZ31 Magnesium Alloy

Friction stir processed (FSPed) AZ31 Mg alloy with fine grains was deformed in tension at room temperature (RT) and 100°C to be compared with the same alloy in the extruded condition with coarse grains. Results indicate that texture affects the deformation behavior of AZ31 alloy more than grain refinement does. The work hardening rate of the FSPed alloy shows a prolonged ascending stage at RTand a plateau stage at 100°C, while that of the extruded alloy keeps dropping rapidly. The above discrepancy between the tensile properties of the two test materials arises since basal slip and f10� 12g tension twin of low critical resolved shear stress are favored by the FSPed texture but not by the extruded texture. 100°C heating allows basal slip and f10�g tension twin to contribute significant improvement to tensile ductility in the FSPed alloy, but hastens the instability in plastic strain of the extruded alloy. The work hardening characteristics and deformation properties of the FSPed specimen in tension at RT and 100°C will be compared further in this paper to examine the different tensile behaviors at these two temperatures. [doi:10.2320/matertrans.M2012081]

Anti-cholinergic effect of singulair on isolated rat’s tracheal smooth muscle

Singulair (Montelukast) is a potent and selective leukotriene D(4) receptor antagonist, often used in treating inflammatory conditions of the respiratory system such as allergic rhinitis and asthma. However, the effects of singulair given intratracheally have rarely been well explored. To verify the effect of singulair, which acts on the tracheal smooth muscle directly in vitro. We used our preparation to test the effects of singulair on isolated rat's tracheal smooth muscle. The following assessments of singulair were performed: (1) effect on the tracheal smooth muscle resting tension, (2) effect on contraction caused by 10(-6) M methacholine as a parasympathetic mimetic, and (3) effect of the drugs on electrically induced tracheal smooth muscle contractions. The results indicated that the addition of methacholine to the incubation medium caused the trachea to contract in a dose-dependent manner. Addition of singulair at doses of 10(-5) M or above elicited a significant relaxation response to 10(-6) M methacholine-induced contraction. Singulair could not inhibit electrical field stimulation-induced spike contraction. It also had a minimal effect on the basal tension of trachea as the concentration increased. This study showed that the high concentrations of singulair also had an anti-cholinergic effect for relieving symptoms of asthma.

Effects of Bupivacaine on the Isolated Rat Tracheal Smooth Muscle

Background Bupivacaine is an amide-linked local anesthetic that has been used in subcutaneous infiltration, epidural and peripheral nerve block for surgery over three decades. Several studies have suggested that inhaled local anesthetics can reduce respiratory reflexes. To assess the direct actions of bupivacaine on airway smooth muscle we used our preparation to examine the effectiveness of bupivacaine on isolated rat trachea. Methods A 5-mm-long portion of rat trachea was mounted in Krebs solution at 37 °C. Changes in tracheal contractility in response to a parasympathetic mimetic agent and electrical stimulation were measured using a transducer connected to a Pentium III computer equipped with polygraphy software. The following assessments were done: (1) effect of bupivacaine on the resting tension of tracheal smooth muscle; (2) effect of bupivacaine on contraction caused by exposure to 10 −6 M methacholine as a parasympathetic mimetic; (3) effect of bupivacaine on electrically induced contraction of tracheal smooth muscle. Results Bupivacaine had a negligible effect on the basal tracheal tension as the concentration increased. Bupivacaine inhibited the tracheal smooth muscle contraction in response to methacholine and electrical stimulation in a concentration-dependent manner. Conclusion The study indicates that bupivacaine might inhibit cholinergic neurotransmission and have an anti-spasmic effect on the trachea, in that it inhibited the contractions elicited by electrical field stimulation and a depolarizing agent.

Evidence that endogenous hydrogen sulfide exerts an excitatory effect on gastric motility in mice

The present study was designed to investigate the effect of endogenous hydrogen sulfide (H 2S) on gastric motility in mice. Western blotting and immunocytochemistry were used to determine expression levels of the H 2S-producing enzymes cystathionine-β-synthase (CBS) and cystathionine-γ-lyase (CSE) in gastric tissues and cultured smooth muscle cells. Physiological and intracellular recordings and the whole-cell patch clamp technique were used to evaluate the effect of H 2S on the mechanical and electrical activities in muscle strips and in isolated smooth muscle cells, respectively. The results showed that CBS and CSE were expressed in mouse gastric smooth muscle. NaHS, a H 2S donor, inhibited the amplitude and frequency of spontaneous contraction at high concentrations (> 200 μM). However, NaHS at low concentrations (< 100 μM) enhanced the basal tension and increased the contractile amplitude of muscle strips. This excitatory effect was not altered by the blockade of the enteric nerve with TTX, but was abolished by tetraethylammonium (TEA) or 4-aminopyridine (4-AP). Aminooxyacetic acid (AOA), but not propargylglycine (PAG), caused a concentration-dependent inhibition of spontaneous contraction. This effect was restored by l-cysteine and NaHS. In addition, NaHS at low concentrations (< 100 μM) produced a depolarization of the membrane potential, whereas AOA hyperpolarized the membrane potential and decreased the amplitude of slow waves. Furthermore, AOA increased whole-cell delayed rectifier K + current ( I K(V)). These findings suggest that endogenous H 2S appears to be an excitatory gaseous mediator during physiological regulation of gastric motility and this excitable effect is mediated by depolarization of the membrane potential via inhibition of I K(V).

The Effects of Wild Ginger (Costus speciosus (Koen) Smith) Rhizome Extract and Diosgenin on Rat Uterine Contractions

The aim of this study was to investigate the effects of wild ginger (Costus speciosus (Koen) Smith, Costaceae) rhizome extract on uterine contractility. We particularly examined the effects on spontaneous phasic contractions and the mechanisms whereby it exerts its effects. Wild ginger rhizomes were ethanolic extracted and their constituents analyzed. Isometric force was measured in strips of longitudinal myometrium and the effects of the extract studied. The extract (10 mg/100 mL) increased spontaneous contractions. The amplitude and frequency of the phasic contraction were significantly increased along with basal tension. Force produced in the presence of the extract was abolished by inhibition of l-type calcium channels or myosin light chain kinase (MLCK). The actions of the extract were not blocked by the estrogen receptor blocker, fulvestrant. Although significant amounts of diosgenin were present in the extract, we found that, depending upon its concentration, diosgenin had either no effect or was inhibitory on force. Interestingly, the extract induced significant amounts of force in the absence of extracellular calcium, which could be blocked by inhibition of the sarcoplasmic reticulum calcium-ATPase (SERCA), but not fulvestrant. We conclude that wild ginger rhizome extract stimulates phasic activity in rat uterus. Our data suggest that the uterotonic effect is due to nonestrogenic effects and not those of diosgenin. Wild ginger was able to increase contraction via calcium entry on l-type calcium channels and sarcoplasmic reticulum (SR) calcium release. We suggest that wild ginger rhizome extract may be a useful uterine stimulant.

ATP causes postjunctional potentiation of noradrenergic contractions in the portal vein of guinea-pig and rat.

Superfusion of the portal vein of rat and guinea-pig with Krebs' solution maintained at 25 degrees C greatly inhibits spontaneous contractions of the preparations and allows contractile responses to ATP and noradrenaline to be measured accurately. Under these conditions, continuous superfusion with ATP (10(-5) M), a concentration which had no effect on either basal tension or spontaneous activity, caused a significant shift to the left of the concentration-response curve to exogenous noradrenaline in both tissues. The mechanism of this potentiation induced by ATP may differ in the two tissues since in the rat portal vein potentiation appeared to be rapidly reversed by superfusing with ATP-free solution, whereas in the guinea-pig portal vein a further concentration-response curve to noradrenaline, in the absence of ATP, was still significantly shifted to the left compared with the control curve. However, potentiation in the rat portal vein may have had a longer duration than is suggested by the results since control concentration-response curves to noradrenaline in this tissue showed a progressive shift to the right which, although not significant, is likely to have affected the apparent time course of potentiation. It is concluded that ATP can potentiate contractions to exogenous noradrenaline in the portal vein of rat and guinea-pig via an, as yet, unidentified postjunctional mechanism.

Phasic activity of urinary bladder smooth muscle in the streptozotocin-induced diabetic rat: Effect of potassium channel modulators

Increased phasic activity in the bladder smooth muscle of animal models and patients with detrusor overactivity has been suggested to underlie the pathophysiology of overactive bladder. Potassium (K+) channels are key regulators of bladder smooth muscle tone and thus may play a role in this altered phasic activity. In this study the effects of K+ channel modulators on the phasic activity of bladder strips from the streptozotocin-induced diabetic rat model of bladder dysfunction were investigated. Bladder strips from rats 1week following streptozotocin administration and age-matched controls were mounted in tissue baths at 37°C and the effects of K+ channel modulators on resting basal tension or phasic activity induced by a low concentration of carbachol (0.5μM) were investigated. Activation of BKCa channels by NS1619 had a minor inhibitory effect on carbachol-induced phasic activity of bladder strips from control and diabetic rats, and significantly inhibited amplitude only at 30μM. Activation of KATP channels by cromakalim inhibited the frequency of carbachol-induced phasic activity of bladder strips, although strips from diabetic rats showed a trend towards being less sensitive to cromakalim. The BKCa channel blocker iberiotoxin was able to induce phasic activity in resting tissues, with diabetic bladder strips demonstrating significantly enhanced phasic activity compared to controls. In contrast, inhibition of SKCa and KATP channels did not induce phasic activity in resting tissues. In conclusion, responses of diabetic rat bladder to BKCa and KATP channel modulators are altered, suggesting altered function and/or expression of channels which may contribute to bladder dysfunction in this model.

Age‐related Changes in Nitric Oxide‐mediated Vasodilation of Cerebral Arteries from Newborn Piglets

This study analyses the age-related changes on the vasodilation mediated by endothelial nitric oxide (NO) in rings of cerebral anterior and communicating arteries from 1–3- (Group 1) or 7–10- (Group 2) day-old piglets. Rings were contracted with phenylephrine (10−5 M) in the presence of indomethacin (10−4 M); then, concentration-response curves to bradykinin (10−10–10−6 M) and to exogenous NO (10−9–10−5 M) were performed. The incubation of segments with L-arginine (10−4 M) or NG-nitro-L-arginine methylester (L-NAME; 3 times 10−5 M) decreased and increased, respectively, the phenylephrine-induced contraction; L-NAME enhanced the basal tension also. Bradykinin relaxation was greater in Group 1 than in Group 2. In Group 1, the incubation with L-arginine did not increase bradykinin relaxation, but incubation with L-NAME abolished it. In Group 2, L-Arginine augmented and L-NAME reduced, but did not abolish, bradykinin relaxation. Vasodilatory response to exogenous NO was higher in Group 1 than in Group 2. We conclude that there is a basal and stimulated NO formation in endothelial cells of newborn piglet cerebral arteries, that the stimulated NO production and the sensitivity to NO are higher in vessels from 1–3-day-old piglets, and that L-arginine supplementation can increase the NO production in the vasculature of 7–10-day-old piglets to overcome its lowest sensitivity to NO.

Renal- and calcium-dependent vascular effects of Polybia paulista wasp venom

In the present study, the effects of Polybia paulista venom (PPV) on renal and vascular tissues were investigated. Isolated kidneys perfused with PPV (1 and 3 μg/mL) had increased perfusion pressure, renal vascular resistance, urinary flow, and glomerular filtration rate; and reduced sodium tubular transport. Histological evaluation demonstrated deposits of proteins in Bowman's space and tubular lumen, and focal areas of necrosis. The venom promoted a cytotoxic effect on Madin-Darby canine kidney (MDCK) cells. A significant increase in lactic dehydrogenase levels was observed in response to venom exposure. In isolated mesenteric vascular beds, pressure and vascular resistance augmented in a dose-dependent manner. PPV increased the contractility of aortic rings maintained under basal tension. This contractile response was inhibited when preparations were maintained in Ca2+-free medium. Likewise, verapamil, a voltage-gated calcium channel blocker, also inhibited the contractile response. In this study, phentolamine, a blocker of α-adrenergic receptor blocker, significantly reduced the contractile effect of PPV in the aortic ring. In conclusion, PPV produced nephrotoxicity, which suggests a direct effect on necrotic cellular death in renal tubule cells. The vascular contractile effect of PPV appears to involve calcium influx through voltage-gated calcium channels via adrenergic regulation.

Effects of reactive oxygen species and neutrophils on endothelium-dependent relaxation of rat thoracic aorta.

Reactive oxygen species (ROS) are produced in different metabolic processes including the respiratory burst of neutrophils accompanying local inflammation. The aim of this study was to analyze the effects of N-formyl-methionyl-leucyl-phenylalanine (FMLP)-activated neutrophils, isolated from the guinea pig peritoneal cavity, on isolated rings of a large (conduit) artery, the rat thoracic aorta. FMLP-activated neutrophils enhanced the basal tension increased by α1-adrenergic stimulation. In phenylephrine-precontracted aortae, they elicited marked contraction, while in noradrenaline-precontracted rat aortal rings they caused a biphasic response (contraction-relaxation). To eliminate interaction of activated neutrophils with catecholamines, in the subsequent experiments the basal tension was increased by KCl-induced depolarization. Activated neutrophils evoked a low-amplitude biphasic response (relaxation-contraction) on the KCl-induced contraction. Not only the acetylcholine- and A23187-induced relaxations but also the catalase sensitive hydrogen peroxide (H2O2) elicited contractions were endothelium-dependent. Even though the acetylcholine-induced relaxation was changed by activated neutrophils and by the ROS studied, their effects differed significantly, yet none of them did eliminate fully the endothelium-dependent acetylcholine relaxation. The effect of activated neutrophils resembled the effect of superoxide anion radical (O2 •–) produced by xanthine/xanthine oxidase (X/XO) and differed from the inhibitory effects of Fe2SO4/H2O2-produced hydroxyl radical (•OH) and H2O2. Thus O2 •– produced either by activated neutrophils or X/XO affected much less the endothelium-dependent acetylcholine-activated relaxation mechanisms than did •OH and H2O2. In the large (conduit) artery, the effects of activated neutrophils and various ROS (O2 •–, •OH and H2O2) seem to be more dependent on muscle tension than on endothelial mechanisms.

Effects of Cromolyn Sodium on Isolated Rat's Trachea

Cromolyn sodium (cromolyn) effectively inhibits both antigen- and exercise-induced asthma when used as an aerosol. Intranasal cromolyn is also recommended for preventing and treating allergic rhinitis. By inhibiting the degranulation of sensitized mast cells, cromolyn reduces the release of mediators that trigger inflammation and the allergic response. The precise pharmacologic activity of cromolyn has not been fully elucidated. This study evaluated the effect of cromolyn on isolated rat's trachea. The following assessments of cromolyn were performed: (1) effect on tracheal resting tension, (2) effect on contraction caused by 10−6 M of methacholine as a parasympathetic mimetic, and (3) effect of the drug on electrically induced tracheal contractions. The results indicated cromolyn could inhibit electrical field stimulation-induced spike contraction when the preparation was increased to 10−4M. Adding cromolyn at doses of ≥10−8 M did not elicit a relaxation or contraction response to 10−6 M of methacholine-induced contraction. It alone had a minimal effect on the basal tension of the trachea as the concentration increased. This study indicates cromolyn had no cholinergic or anticholinergic effect and high concentrations of cromolyn might actually inhibit parasympathetic function of the trachea. Inhibiting parasympathetic function of the trachea through stabilizing the presynaptic nerve by cromolyn may be responsible for protecting patients against antigen- and exercise-induced asthma.

Prolonged mechanical stretch is associated with upregulation of hypoxia-inducible factors and reduced contraction in rat inferior vena cava

Decreased venous tone and vein wall dilation may contribute to varicose vein formation. We have shown that prolonged vein wall stretch is associated with upregulation of matrix metalloproteases (MMPs) and decreased contraction. Because hypoxia-inducible factors (HIFs) expression also increases with mechanical stretch, this study tested whether upregulation of HIFs is an intermediary mechanism linking prolonged vein wall stretch to the changes in MMP expression and venous contraction. Segments of rat inferior vena cava (IVC) were suspended in tissue bath under 0.5-g basal tension for 1 hour, and a control contraction to phenylephrine (PHE, 10(-5)M) and KCl (96 mM) was elicited. The veins were then exposed to prolonged 18 hours of tension at 0.5 g, 2 g, 2 g plus HIF inhibitor U0126 (10(-5)M), 17-[2-(dimethylamino)ethyl] amino-17-desmethoxygeldanamycin (17-DMAG, 10(-5)M), or echinomycin (10(-6)M), or 2 g plus dimethyloxallyl glycine (DMOG; 10(-4)M), a prolyl-hydroxylase inhibitor that stabilizes HIF. The fold-change in PHE and KCl contraction was compared with the control contraction at 0.5-g tension for 1 hour. Vein tissue homogenates were analyzed for HIF-1α, HIF-2α, MMP-2, and MMP-9 messenger RNA (mRNA) and protein amount using real-time reverse transcription polymerase chain reaction and Western blots. Compared with control IVC contraction at 0.5-g tension for 1 hour, the PHE and KCl contraction after prolonged 0.5-g tension was 2.0 ± 0.35 and 1.1 ± 0.06, respectively. Vein contraction to PHE and KCl after prolonged 2-g tension was significantly reduced (0.87 ± 0.13 and 0.72 ± 0.05, respectively). PHE-induced contraction was restored in IVC exposed to prolonged 2-g tension plus the HIF inhibitor U0126 (1.38 ± 0.15) or echinomycin (1.99 ± 0.40). U0126 and echinomycin also restored KCl-induced contraction in IVC exposed to prolonged 2-g tension (1.14 ± 0.05 and 1.11 ± 0.15, respectively). Treatment with DMOG further reduced PHE- and KCl-induced contraction in veins subjected to prolonged 2-g tension (0.47 ± 0.06 and 0.57 ± 0.01, respectively). HIF-1α and HIF-2α mRNA were overexpressed in IVC exposed to prolonged 2-g tension, and the overexpression was reversed by U0126. The overexpression of HIF-1α and HIF-2α in stretched IVC was associated with increased MMP-2 and MMP-9 mRNA. The protein amount of HIF-1α, HIF-2α, MMP-2, and MMP-9 was also increased in IVC exposed to prolonged 2-g wall tension. Prolonged increases in vein wall tension are associated with overexpression of HIF-1α and HIF-2α, increased MMP-2 and MMP-9 expression, and reduced venous contraction in rat IVC. Together with our report that MMP-2 and MMP-9 inhibit IVC contraction, the data suggest that increased vein wall tension induces HIF overexpression and causes an increase in MMP expression and reduction of venous contraction, leading to progressive venous dilation and varicose vein formation.

Contribution of Rho kinase to the early phase of the calcium-contraction coupling in airway smooth muscle

We investigated theoretically and experimentally the role of Rho kinase (RhoK) in Ca(2+)-contraction coupling in rat airways. Isometric contraction was measured on tracheal, extrapulmonary and intrapulmonary bronchial rings. Intracellular [Ca(2+)] was recorded in freshly isolated tracheal myocytes. Stimulation by carbachol (0.3 and 10 μm) and 50 mm external KCl induced a short-time, Hill-shaped contraction obtained within 90 s, followed by a sustained or an additional delayed contraction. Responses of [Ca(2+)](i) to acetylcholine consisted in a fast peak followed by a plateau and, in 42% of the cells, superimposed Ca(2+) oscillations. The RhoK inhibitor Y27632 (10 μm) did not alter the [Ca(2+)](i) response. Whatever the agonist, Y27632 did not modify the basal tension but decreased the amplitude of the short-duration response, without altering the additional delayed contraction. The Myosin Light Chain Phosphatase (MLCP) inhibitor calyculin A increased the basal tension and abolished the effect of RhoK. KN93 (Ca(2+)-calmodulin-dependent protein kinase II inhibitor) and DIDS (inhibitor of Ca(2+)-activated Cl(-) channels) had no influence on the RhoK effect. We built a theoretical model of Ca(2+)-dependent active/inactive RhoK ratio and subsequent RhoK-dependent MLCP inactivation, which was further coupled with a four-state model of the contractile apparatus and Ca(2+)-dependent MLCK activation. The model explains the time course of the short-duration contraction and the role of RhoK by Ca(2+)-dependent activation of MLCK and RhoK, which inactivates MLCP. Oscillatory and non-oscillatory [Ca(2+)](i) responses result in a non-oscillatory contraction, the amplitude of which is encoded by the plateau value and oscillation frequency. In conclusion, Ca(2+)-dependent but CaMK II-independent RhoK activation contributes to the early phase of the contractile response via MLCP inhibition.

Efectos de ruda ssp sobre la actividad del musculo liso gastrointestinal aislado de rata

Ruta graveolens L. and Ruta chalepensis L. are plants used in folkloric medicine as antispasmodics, digestive and for intestinal gases. Animals used as experimental model were rates of the Wistar line, adult females, clinically healthy and with a weight average of 250 grams. We used strips of stomach and duodenum. Each one of the segments mounted on two stirrups in a bath of organ isolated with Ringer-lactate solution, at 37° C , pH: 7.3- 7.4, and bubbled with 95% O2, 5% CO2. One of the stirrups was connected vertically to the bottom of the bath and the other to a transducer of tension connected to a Beckman polygraph. We applied 500 mgs of basal tension. After the stabilization, the ethanolic extract of Ruta ssp was added in increasing doses.At 50?l/ml the tone lower 23% in small intestine and lower 27% in stomach. However at 100 ?l/ml the tone lower 32% and 35% respectively. In the other parameters the amplitude decrease 50% in the stomach at dose of 5 ?l/ml while in the small intestine the amplitude lower 60%. With 10 ?l/ml the amplitude change in both organs ( 96% in small intestine, and 75% in stomach).The frequency changes in both organs ( 32% in small intestine, and 50% in stomach) at 10 ?l/ml Rue showed decreased effects on isolated small intestine and stomach were is dose dependent, maybe we were demonstrated the effects digestive of Ruta.

Azelastine nasal spray inhibiting parasympathetic function of tracheal smooth muscle

Azelastine hydrochloride is a histamine receptor-1 (H(1)) antagonist with anti-inflammatory properties that is available in the United States as Astelin Nasal Spray for rhinitis patients who are suffering from sneezing and rhinorrhea. The effect of H(1) antagonists on nasal mucosa in vivo is well known; however, the effect of the drug on tracheal smooth muscle has rarely been explored. During administration via oral intake or inhalation of the H(1) antagonist for nasal symptoms, it might affect the trachea. We examined the effectiveness of azelastine on isolated rat tracheal smooth muscle by testing: 1) the effect on tracheal smooth muscle resting tension; 2) the effect on contraction caused by 10(-6) M methacholine as a parasympathetic mimetic; and 3) the effect on electrically induced tracheal smooth muscle contractions. The results indicated that addition of methacholine to the incubation medium caused the trachea to contract in a dose-dependent manner. Addition of azelastine at doses of 10(-5) M or above elicited a significant relaxation response to 10(-6) M methacholine-induced contraction. Azelastine could inhibit electrical field stimulation-induced spike contraction. It alone had a minimal effect on the basal tension of trachea as the concentration increased. This study indicated that high concentrations of azelastine might actually inhibit parasympathetic function of the trachea. Azelastine might reduce asthma attacks in rhinitis patients because it could inhibit parasympathetic function and reduce methacholine-induced contraction of tracheal smooth muscle.

Mechanisms involved in the effects of endothelin-1 in pig prostatic small arteries

Since endothelin-1 (ET-1) is involved in prostatic disorders, the current study investigated the mechanisms underlying the ET-1-induced effects in pig prostatic small arteries. The experiments were performed in rings mounted in microvascular myographs containing physiological saline solution at 37 oC for isometric force recordings. On basal tension, ET-1 (0.1–30 nM) evoked concentration-dependent contractions, which were enhanced by endothelium removal. ET-1 contractions were inhibited by blockade of endothelin ET A and ET B receptors, extracellular Ca 2+ removal and blockade of voltage-dependent (L-type)- and non-voltage-dependent-Ca 2+ channels. On endothelium intact rings precontracted with noradrenaline, the ET B endothelin receptor agonist BQ3020 promoted a concentration-dependent relaxation which was reduced by blockade of ET B receptors, nitric oxide synthase, guanylyl cyclase and prostanoids synthesis. Endothelium removal abolished its relaxant response and unmasked a BQ3020-induced contraction. Tetraethylammonium and 4-aminopyridine, blockers of non-selective K + channels and voltage-dependent K + (Kv) channels, respectively, inhibited the relaxations to BQ3020. Iberiotoxin, apamin and glibenclamide, blockers of large and small Ca 2+-activated- and ATP-dependent- K + channels, respectively, failed to modify these responses. These data suggest that ET-1 promotes contraction of pig prostatic small arteries by activating vascular smooth muscle contractile endothelin ET A and ET B receptors coupled to extracellular Ca 2+ entry, via voltage-dependent (L-type)- and non-voltage-dependent Ca 2+ channels, also being due to intracellular Ca 2+ mobilization. In addition, a population of endothelial ET B receptors mediates vasorelaxation via NO-cGMP pathway, vasodilator cyclooxygenase product(s) and Kv channels.

190 STIMULATION OF β3-ADRENOCEPTORS INHIBITS EFS-INDUCED CONTRACTIONS OF HUMAN ISOLATED URINARY BLADDER

You have accessJournal of UrologyBladder and Urethra: Anatomy, Physiology and Pharmacology I1 Apr 2010190 STIMULATION OF β3-ADRENOCEPTORS INHIBITS EFS-INDUCED CONTRACTIONS OF HUMAN ISOLATED URINARY BLADDER Moèz Rekik, Céline Rouget, Philippe Camparo, Henry Botto, Philippe Lluel, Timothy Westfall, and Stefano Palea Moèz RekikMoèz Rekik Toulouse, France More articles by this author , Céline RougetCéline Rouget Toulouse, France More articles by this author , Philippe CamparoPhilippe Camparo Suresnes, France More articles by this author , Henry BottoHenry Botto Suresnes, France More articles by this author , Philippe LluelPhilippe Lluel Toulouse, France More articles by this author , Timothy WestfallTimothy Westfall Toulouse, France More articles by this author , and Stefano PaleaStefano Palea Toulouse, France More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2010.02.246AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Activation of β-adrenoceptors (β-AR) has been shown to produce relaxation of pre-contracted urinary bladder smooth muscle suggesting that β-AR agonism could promote relaxation of basal tension during urine storage. However, very little is known about how β-AR agonism affects neuronal efferent activity. The aim of the present study was to investigate the effect of β-AR agonism on neurogenic contractions of human isolated detrusor and the β-ARs involved. METHODS Detrusor smooth muscle strips devoid of urothelium were obtained from 11 patients (72 ± 3 years old, 10 male and 1 female) undergoing cystectomy due to bladder cancer. Strips were mounted under 1 g of initial tension and electrical field stimulation (EFS) applied using the following parameters: maximal current, frequency of 10 Hz, square pulses of 0.1 ms, trains of 5 s every min. L748,337 (β3-AR antagonist at 0,3, 1 and 3 μM), ICI118,551 (β2-AR antagonist at 30 and 100 nM) or vehicle were added, followed 20 min later by a cumulative concentration-response curves to a β-AR agonists (isoproterenol, L755,507 or CL316,243). Responses are expressed as % variation from basal EFS-induced contractions. RESULTS Isoproterenol concentration-dependently inhibited TTX-sensitive EFS-induced contractions of human bladder smooth muscle strips with an EC50 value of 0.19 ± 0.11 μM and an Emax value of 66.6 ± 3.9% at 30 μM. In contrast L755,507 and CL316,243 decreased EFS-induced contractions by only 30.0 ± 8.9 % at 10 μM and 20.8 ± 3.5 % at 30 μM, respectively. The β2-AR antagonist, ICI118,551 at 30 and 100 nM had no effect on the response to isoproterenol. However, the β3-AR antagonist L748,337 induced a concentration-dependent rightward shift of the isoproterenol response without affecting the maximal effect. Schild plot analysis revealed a pA2 value of 7.37 and a slope of 1.07 ± 0.19 indicating competitive antagonism. CONCLUSIONS While it is well known that stimulation of β-ARs produces relaxation of basal tension and carbachol pre-contracted detrusor muscle, there are very few reports on the effect of β-AR agonists on EFS induced-responses. Here, we clearly demonstrate that isoproterenol is able to significantly inhibit EFS-induced contractions of human detrusor muscle through activation of β3-ARs. It remains to be determined if this effect is pre- or post-junctional, however the results suggest that in addition to producing relaxation of basal tension during urine storage, β3-AR agonism might inhibit neuronally mediated non-voiding and voiding contractions. © 2010 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 183Issue 4SApril 2010Page: e75-e76 Advertisement Copyright & Permissions© 2010 by American Urological Association Education and Research, Inc.MetricsAuthor Information Moèz Rekik Toulouse, France More articles by this author Céline Rouget Toulouse, France More articles by this author Philippe Camparo Suresnes, France More articles by this author Henry Botto Suresnes, France More articles by this author Philippe Lluel Toulouse, France More articles by this author Timothy Westfall Toulouse, France More articles by this author Stefano Palea Toulouse, France More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

The Effects of Pomegranate Seed Extract and β-Sitosterol on Rat Uterine Contractions

The aim of this study was to investigate the effects of pomegranate (Punica granatum L., Punicaceae) seed extract on uterine contractility. Pomegranate seeds were methanolic extracted and their constituents analyzed using gas chromatography and mass spectrometry. Isometric force was measured in strips of longitudinal rat myometrium and the effects of pomegranate seed extract studied. We found beta-sitosterol to be the main constituent of the extract (16%) and its effects were also investigated. Pomegranate seed extract and beta-sitosterol increased spontaneous contractions in a concentration-dependent manner with a maximum effect at 250 mg/100 mL and 1 mg/100 mL, respectively. The amplitude and frequency of the phasic contraction were significantly increased along with basal tension. The effects of pomegranate seed extract were very similar to those of beta-sitosterol. Force produced in the presence of pomegranate seed extract was abolished by the inhibition of L-type calcium channels or myosin light chain kinase (MLCK). Contractions were not potentiated by pomegranate extract following the inhibition of K channels or inhibition of the sarcoplasmic reticulum calcium ATPase (SERCA). The actions of beta-sitosterol and the extract were not blocked by the estrogen receptor blocker, fulvestrant. We conclude that pomegranate seed extract is a potent stimulator of phasic activity in rat uterus. Our data suggest that the uterotonic effect is due to nonestrogenic effects of beta-sitosterol acting to inhibit K channels and SERCA and thereby increasing contraction via calcium entry on L-type calcium channels and MLCK. We suggest that pomegranate extract and beta-sitosterol may be a useful uterine stimulant.