ACh-induced Contraction Research Articles

Effects of STW 5 and STW 6 on rat ileal and colonic preparations: A comparative study

STW 5 (Iberogast®) is a fixed combination of nine plant extract with Iberis amara L. (STW 6) as its main component. It is successfully used for treatment of functional dyspepsia or irritable bowel syndrome (IBS). Because clinical data suggest an inflammatory etiology of IBS the influence of STW 5 and STW 6 on tone and acetylcholine (ACh)-induced contractions of intact and inflamed intestinal preparations was examined. We used 1–1.5cm long ileum and colon preparations of male Wistar rats to analyze region specific differences. Inflammation was induced by intraluminal instillation of 2,4,6-trinitrobenzene sulfonic acid (TNBS, 10mM, 30min). STW 5 (64–512µg/ml) concentration dependently reduced the tone and decreased ACh-induced contractions of intact ileal and colonic preparations. STW 6 in equivalent concentrations (3–24.1µg/ml) neither affected tone nor contractility. TNBS-induced inflammation was accompanied by a significantly reduced ACh contractions and morphological disturbances. Co-incubation of TNBS with STW 5 (512µg/ml) or STW 6 (24.1µg/ml) partially normalized the TNBS-induced attenuation of ACh-induced contractions andmorphological damage in ileum preparations, whereas in inflamed colon segments only the co-incubation of TNBS with STW 6 in a high concentration (24.1µg/ml) revealed similar effects.

Relaxant effects of Schisandra chinensis and its major lignans on agonists-induced contraction in guinea pig ileum

In this study, the herbal extracts of Schisandra chinensis were demonstrated to inhibit the contractions induced by acetylcholine (ACh) and serotonin (5-HT) in guinea pig ileum, and the 95% ethanol extract was more effective than the aqueous extract. Analysis with High Performance Liquid Chromatography (HPLC) indicated that schisandrin, schisandrol B, schisandrin A and schisandrin B were the major lignans of Schisandra chinensis, and the ethanol extract contained higher amount of these lignans than the aqueous extract. All four lignans inhibited the contractile responses to ACh, with EC 20 values ranging from 2.2 ± 0.4 μM (schisandrin A) to 13.2 ± 4.7 μM (schisandrin). The effectiveness of these compounds in relaxing the 5-HT-induced contraction was observed with a similar magnitude. Receptor binding assay indicated that Schisandra lignans did not show significant antagonistic effect on muscarinic M3 receptor. In Ca 2+-free preparations primed with ACh or KCl, schisandrin A (50 μM) attenuated the contractile responses to cumulative addition of CaCl 2 by 37%. In addition, schisandrin A also concentration-dependently inhibited ACh-induced contractions in Ca 2+-free buffer. This study demonstrates that Schisandra chinensis exhibited relaxant effects on agonist-induced contraction in guinea pig ileum, with schisandrin, schisandrol B, schisandrin A and schisandrin B being the major active ingredients. The antispasmodic action of schisandrin A involved inhibitions on both Ca 2+ influx through L-type Ca 2+ channels and intracellular Ca 2+ mobilization, rather than specific antagonism of cholinergic muscarinic receptors.

The effect of boric acid on acethylcholine, bethanechol and potasssium‐evoked responses on ileum of rat

1 The aim of this study was to clarify the effect of boric acid on contractions of rat isolated ileum. 2 Contractile responses expressed as Emax and pD2 for acetylcholine (10(-3)-10(-8) m, Ach), bethanechol (10(-3)-10(-8) m) and potassium (10-80 × 10(-3) m, KCl) were determined in the absence and presence of boric acid (10(-3); 5 × 10(-4); 10(-4) m). 3 The contractile response to Ach in the presence of verapamil (10(-6) or 10(-8) m) or in calcium-free Tyrode's solution was also determined in the absence and presence of boric acid. 4 Boric acid did not affect the contractile response to Ach, bethanechol or KCl. Single or cumulative treatment of boric acid did not affect ileum muscle contraction evoked by KCl. The atropine-resistant component of Ach-induced contraction and 4-diphenyl-acetoxy-N-methyl-piperidine methiodide-resistant component of bethanechol-induced contraction were not inhibited by boric acid (10(-3) m). The contractile response to Ach was reduced in calcium-free Tyrode's solution, and the contractile response was not affected by (10(-8) m). The addition of boric acid (10(-3) m) in combination with verapamil (10(-8) m) did not significantly affect the contractile response to Ach. 5 In conclusion, boric acid does not affect contractions induced by Ach, bethanechol or potassium in rat isolated ileum.

Role of TLR4 and MAPK in the local effect of LPS on intestinal contractility

Lipopolysaccharide (LPS) has been shown to alter intestinal contractility. Toll-like receptor 4 (TLR4), K(+) channels and mitogen-activated protein kinases (MAPKs) have been proposed to be involved in the mechanism of action of LPS. The aim of this study was to determine the role of TLR4, K(+) channels and MAPKs (p38, JNK and MEK1/2) in the local effect of LPS on the acetylcholine (ACh)-induced contractions in rabbit small intestine in vitro. Segments of rabbit duodenum were suspended in the direction of longitudinal or circular smooth muscle fibres in a thermostatically controlled organ bath. LPS (0.3 µg/ml) reduced the contractions induced by ACh (100 µm) in the longitudinal and circular smooth muscle of the duodenum after 90 min of incubation. Polymyxin (TLR4 inhibitor), SB203580 (p38 MAPK inhibitor), SP600125 (JNK1/2 inhibitor) and U0126 (MEK1/2 inhibitor) antagonized the effects of the LPS on ACh-induced contractions in duodenal smooth muscle. Incubation with the blockers of K(+) channels, TEA, apamin, charybdotoxin, iberiotoxin, glibenclamide or quinine, did not reverse the effect of LPS on ACh-induced contractions. These results suggest that the effect of LPS on ACh-induced contractions in the rabbit duodenum might be mediated by TLR4 and p38, JNK1/2 and MEK1/2 MAPKs.

Gβγ‐activated phosphoinositide 3‐kinase γ regulates airway smooth muscle contraction by modulating calcium oscillations

Airway smooth muscle (ASM) contractile hyper‐responsiveness plays a critical role in asthma. Muscarinic receptors in ASM convey bronchoconstrictor signals by dissociating G‐proteins into active Gα and Gβγ subunits. We found that expression of a Gβγ scavenger peptide suppressed acetylcholine (ACh)‐stimulated mouse ASM cell contraction. Gβγ plays prominent roles in signal transduction through its downstream effectors including PI3Kγ. Immunohistochemistry staining and Western blot indicated expression of PI3Kγ in ASM cells of mouse trachea, lung and cultured ASM cells. Inhibitors of PI3Kγ, but not PI3Kα, β, or δ, attenuated ACh‐stimulated airway contraction of mouse lung slices. It is noteworthy that airways in lung slices pretreated with PI3Kγ inhibitors still exhibited an ACh‐induced initial contraction, but the sustained contraction was significantly reduced. Furthermore, PI3Kγ inhibitors or silence of endogenous PI3Kγ by siRNA had a small inhibitory effect on the ACh‐stimulated initial Ca2+ transient in mouse ASM cells but significantly attenuated the sustained Ca2+ oscillations that are critical for sustained airway contraction. Thus, Gβγ/PI3Kγ signaling pathway controls contractility of airways through regulation of Ca2+ oscillations in ASM cells. Targeting this pathway may directly impact airway hyper‐responsiveness associated with asthma. Supported by Nebraska State LB595 and 692.

Study of the reversal effect of NF449 on neuromuscular blockade induced by d-tubocurarine

AimsThe aim of this study was to investigate the mechanism for the reversal effect of NF449 (a suramin analogue) on the neuromuscular block induced by d-tubocurarine (d-TC). Main methodsNerve-stimulated muscle contractions and end-plate potentials were performed in mouse phrenic nerve-diaphragm preparations. Acetylcholine (ACh)-induced muscle contractions were performed in the chick biventer cervicis preparations. Presynaptic nerve terminal waveform recordings were performed in mouse triangularis sterni preparations. Key findingsAmongst the suramin analogues in this study, only the NF449 and suramin were able to reverse the blockade effect produced by d-TC on nerve-stimulated muscle contractions. Each of these suramin analogues (NF007, NF023, NF279 and NF449) alone has no significant effect on the amplitude of nerve-stimulated muscle contractions. NF449 and suramin also showed the antagonising effects on the inhibition of end-plate potentials induced by d-TC. Furthermore, pre-treatment with NF449 can antagonise the inhibition of d-TC in ACh-induced contractions of chick biventer cervicis muscle. NF449 produced a greater rightward shift of the dose–response inhibition curve for d-TC than did suramin. Because other purinergic 2X (P2X) receptor antagonists, NF023 and NF279, do not have the reverse effects on the neuromuscular blockade of d-TC, the effect of NF449 seems irrelevant to inhibition of P2X receptors. SignificanceThese data suggest that NF449 was able to compete with the binding of d-TC on the nicotinic ACh receptors, and the effect of NF449 was more potent than suramin in reducing the inhibition of d-TC. The structure of NF449 may provide useful information for designing potent antidotes against neuromuscular toxins.

M1 and M3 Muscarinic Receptors Mediate Relaxation and Contraction in Canine Nasal Veins

Acetylcholine (ACh) has been shown to induce nasal congestion via vasorelaxation of intranasal posterior collecting veins (PCV) coupled with vasocontraction of extranasal outflow veins (dorsal nasal vein [DNV] and sphenopalatine vein [SPV]). The aim of this study was to characterize the muscarinic receptor subtype(s) involved in ACh-induced relaxation and contraction in canine nasal veins. PCV, DNV, and SPV were isolated from the canine nose. In vitro isometric tension of segments from these veins was monitored to reflect vascular reactivity. ACh concentration-response curve was studied in the presence of muscarinic receptor subtype inhibitors. Immunohistochemical localization of M(1)-M(5) receptor subtypes in the veins was performed. ACh-induced relaxation in PVC was inhibited by pertussis toxin (PTX; inhibitor of G-protein that couples M(2)/M(4) receptors), methoctramine (selective M(2) muscarinic receptor inhibitor), muscarinic toxin 7 (MT-7; selective M(1) muscarinic receptor inhibitor), and 4-diphenylacetoxy-methylpiperidine methiodide (4-DAMP; selective M(3) muscarinic receptor inhibitor). ACh-induced contraction in SPV and DNV was potentiated by PTX and methoctramine but was inhibited by MT-7 and 4-DAMP. Immunohistochemistry confirmed the presence of five muscarinic receptor subtypes in the endothelium of nasal veins, with staining of M(3) > M(1) > M(5) > M(2) = M(4) in PVC but M(2) = M(4) > M(3) > M(1) > M(5) in outflow veins. M(1) and M(3) receptor subtypes were localized in the smooth muscles of both types of veins. The results show that ACh relaxes intranasal veins and contracts extranasal veins primarily via M(1) and M(3) muscarinic receptor subtypes, implying the therapeutic value of M(1)/M(3)-specific or highly selective anticholinergics on nasal congestion.

Activation of Vinculin Induced by Cholinergic Stimulation Regulates Contraction of Tracheal Smooth Muscle Tissue

Vinculin localizes to membrane adhesion junctions where it links actin filaments to the extracellular matrix by binding to the integrin-binding protein talin at its head domain (Vh) and to actin filaments at its tail domain (Vt). Vinculin can assume an inactive (closed) conformation in which Vh and Vt bind to each other, masking the binding sites for actin and talin, and an active (open) conformation in which the binding sites for talin and actin are exposed. We hypothesized that the contractile activation of smooth muscle tissues might regulate the activation of vinculin and thereby contribute to the regulation of contractile tension. Stimulation of tracheal smooth muscle tissues with acetylcholine (ACh) induced the recruitment of vinculin to cell membrane and its interaction with talin and increased the phosphorylation of membrane-localized vinculin at the C-terminal Tyr-1065. Expression of recombinant vinculin head domain peptide (Vh) in smooth muscle tissues, but not the talin-binding deficient mutant head domain, VhA50I, inhibited the ACh-induced recruitment of endogenous vinculin to the membrane and the interaction of vinculin with talin and also inhibited vinculin phosphorylation. Expression of Vh peptide also inhibited ACh-induced smooth muscle contraction and inhibited ACh-induced actin polymerization; however, it did not affect myosin light chain phosphorylation, which is necessary for cross-bridge cycling. Inactivation of RhoA inhibited vinculin activation in response to ACh. We conclude that ACh stimulation regulates vinculin activation in tracheal smooth muscle via RhoA and that vinculin activation contributes to the regulation of active tension by facilitating connections between actin filaments and talin-integrin adhesion complexes and by mediating the initiation of actin polymerization.

Novel expression of a functional glycine receptor chloride channel that attenuates contraction in airway smooth muscle

Airway smooth muscle (ASM) contraction is an important component of the pathophysiology of asthma. Taurine, an agonist of glycine receptor chloride (GlyR Cl(-)) channels, was found to relax contracted ASM, which led us to question whether functional GlyR Cl(-) channels are expressed in ASM. Messenger RNA for β (GLRB), α1 (GLRA1), α2 (GLRA2), and α4 (GLRA4) subunits were found in human (Homo sapiens) and guinea pig (Cavia porcellus) tracheal smooth muscle. Immunoblotting confirmed the protein expression of GLRA1 and GLRB subunits in ASM. Electrical activity of cultured human ASM cells was assessed using a fluorescent potentiometric dye and electrophysiological recordings. Glycine increased current and significantly increased fluorescence in a dose-dependent manner. The GlyR Cl(-) channel antagonist strychnine significantly blocked the effects of glycine on potentiometric fluorescence in ASM cells. Guinea pig airway ring relaxation of ACh-induced contractions by isoproterenol was significantly left-shifted in the presence of glycine. This effect of glycine was blocked by pretreatment with the GlyR Cl(-) channel antagonist strychnine. Glycine treatment during tachykinin- and acetylcholine-induced contractions significantly decreased the maintenance of muscle force compared to control. GlyR Cl(-) channels are expressed on ASM and regulate smooth muscle force and offer a novel target for therapeutic relaxation of ASM.

In vitro effects of Artemisia dracunculus essential oil on ruminal and abomasal smooth muscle in sheep

Recently, essential oils have been substituted for synthetic antibiotics in improving rumen fermentation and animal performance. When ingested, along with their positive properties, some adverse effects may also be observed in animals. This study was primarily aimed at investigating the effects of the essential oils extracted from Artemisia dracunculus (EOAD) on ruminal and abomasal smooth muscle. EOAD was extracted using hydrodistillation. Sixteen compounds, representing 94.06% of the oil, were identified using gas chromatography/mass spectrometry analyses, Hinokitiol (17.47%), estragole (17.28%), pulegone (10.23%), limonene (7.57%), methyl eugenol (7.46%), and bornyl acetate (7.12%) were the major compound in the oil. In the in vitro study, except for isolated ruminal strips, abomasal preparations from 24 healthy sheep, EOAD (0.1–100 μg/mL) evoked a weak spasmogenic effect followed by relaxation, but complete abolishment of spontaneous contraction occurred at the highest dose (1,000 μg/mL) (P < 0.05). EOAD significantly (P < 0.05) inhibited the Ach-induced contraction when tissues were pre-incubated with the highest doses. When animal rations are supplemented with essential oils, a reduced basal tone and an impaired response of the rumen and abomasal muscles to acetylcholine reflect hypotony and subsequently predispose the animals to abomasal displacement, abomasal rotation, and or indigestion.

Antispasmodic effects of eugenol on rat airway smooth muscle

This study was undertaken to assess the effects of eugenol (EUG) on tracheal muscle (TM) and the putative mechanisms underlying these effects. Cumulatively increasing concentrations (1-1000 μm) of EUG did not affect the resting tonus of TM. However, EUG (1-2000 μm) reduced the contractions induced by electrical field stimulation (IC(50) = 842.3 ± 52.7 μm), an effect that was unaltered by either 10 μm montelukast (IC(50) = 816.1 ± 70.1 μm) or 2 μm indomethacin (IC(50) = 693.1 ± 170.8 μm). EUG also completely relaxed the sustained contractile responses to 80 mM K(+) (IC(50) = 597.3 ± 60.6 μm) and 1 μm carbamoylcholine (IC(50) = 571.3 ± 148.8 μm), an effect that was unaltered by indomethacin (2 μm). Under Ca(2+) -free conditions, EUG reduced the ACh-induced contractions (IC(50) = 703.4 ± 256.1 μm), the CaCl₂ -induced contractions in preparations pretreated with 60 μm ACh in the presence of nifedipine, and the Ba(2+) -induced contractions in preparations depolarized with K(+) . In tracheal preparations maintained in Ca(2+) -containing solution, EUG (300-2000 μm) relaxed the contractile response to phorbol dibutyrate (1 μm), an activator of protein kinase C. It is concluded that in TM, EUG induces a myogenic antispasmodic effect (not modulated by arachidonic acid derivatives) either through various mechanisms almost with the same pharmacological potency or via an action on a step common to all of them. These mechanisms seem to include blockade of voltage- and receptor-operated Ca(2+) channels, IP₃ -induced Ca(2+) release from sarcoplasmic reticulum and reduction of the sensitivity of contractile proteins to Ca(2+) .

Antispasmodic effects of essential oil of Pterodon polygalaeflorus and its main constituent β‐caryophyllene on rat isolated ileum

This study investigates the effects of essential oil of Pterodon polygalaeflorus (EOPP) and β-caryophyllene (β-CAR). EOPP and β-CAR relaxed the basal tone of ileum smooth muscle in a concentration-dependent manner (IC(50) s = 394.35 ± 62.12 and 68.65 ± 9.51 μg/mL respectively), an effect that was unaltered by hexamethonium, L-nitroarginine methyl ester or indomethacin. Both EOPP and β-CAR evoked a concentration-dependent relaxation of ileum pre-contracted with KCl with an IC(50) value of 107.78 ± 10.47 and 17.35 ± 0.75 μg/mL, respectively. EOPP and β-CAR inhibited the contractions induced by acetylcholine (ACh) and by KCl. In ileal preparations, the CaCl(2) -induced contractions were reduced by EOPP (300 μg/mL) and β-CAR (100 μg/mL). Furthermore, CaCl(2) -induced contractions were also reduced by EOPP (300 μg/mL) and β-CAR (100 μg/mL) in ileal preparations pretreated with ACh under Ca(2+) -free condition and in the presence of verapamil. EOPP (100 and 300 μg/mL) and β-CAR (30 and 100 μg/mL) reduced the ACh-induced contractions of isolated rat ileum under Ca(2+) -free conditions. In the presence of high KCl and Ca(2+) -free conditions, EOPP (300 μg/mL) and β-CAR (100 μg/mL) reduced the contractions induced by barium. A similar effect was also observed with verapamil. It is concluded that (i) β-CAR is an important constituent involved in the myorelaxant and antispasmodic effects induced by EOPP; (ii) the inhibitory effect on intestinal contractility is myogenic and seems mainly mediated through an intracellular mechanism. However, the ability of EOPP and β-CAR to decrease Ca(2+) influx through cytoplasmic membrane could not be discounted.

Phosphorylated HSP20 modulates the association of thin-filament binding proteins: caldesmon with tropomyosin in colonic smooth muscle

Small heat shock proteins HSP27 and HSP20 have been implicated in regulation of contraction and relaxation in smooth muscle. Activation of PKC-α promotes contraction by phosphorylation of HSP27 whereas activation of PKA promotes relaxation by phosphorylation of HSP20 in colonic smooth muscle cells (CSMC). We propose that the balance between the phosphorylation states of HSP27 and HSP20 represents a molecular signaling switch for contraction and relaxation. This molecular signaling switch acts downstream on a molecular mechanical switch [tropomyosin (TM)] regulating thin-filament dynamics. We have examined the role of phosphorylation state(s) of HSP20 on HSP27-mediated thin-filament regulation in CSMC. CSMC were transfected with different HSP20 phosphomutants. These transfections had no effect on the integrity of actin cytoskeleton. Cells transfected with 16D-HSP20 (phosphomimic) exhibited inhibition of acetylcholine (ACh)-induced contraction whereas cells transfected with 16A-HSP20 (nonphosphorylatable) had no effect on ACh-induced contraction. CSMC transfected with 16D-HSP20 cDNA showed significant decreases in 1) phosphorylation of HSP27 (ser78); 2) phosphorylation of PKC-α (ser657); 3) phosphorylation of TM and CaD (ser789); 4) ACh-induced phosphorylation of myosin light chain; 5) ACh-induced association of TM with HSP27; and 6) ACh-induced dissociation of TM from caldesmon (CaD). We thus propose the crucial physiological relevance of molecular signaling switch (phosphorylation state of HSP27 and HSP20), which dictates 1) the phosphorylation states of TM and CaD and 2) their dissociations from each other.

Inhibition of p38 MAPK-dependent bronchial contraction after ozone by corticosteroids

We determined the role of p38 mitogen-activated protein kinase (MAPK) in the increased airway smooth muscle (ASM) contractile responses following ozone and modulation by corticosteroids. Mice were exposed to air or ozone (3 ppm for 3 h) and isometric contractile responses of bronchial rings to acetylcholine (ACh) were measured using a myograph in the presence of p38 MAPK inhibitor, SB239063 (10⁻⁶ M) or dexamethasone (10⁻⁶ M). Because MAPK phosphatase (MKP)-1 is a negative regulator of p38 MAPK, we also studied these effects in MKP-1(-/-) mice. Bronchial rings from ozone-exposed wild-type and MKP-1(-/-) mice showed increased contractile responses, with a leftward shift of the dose-response curve in MKP-1(-/-) mice. SB239063 inhibited bronchial contraction equally in air- and ozone-exposed C57/BL6 and MKP-1(-/-) mice. Dexamethasone inhibited ACh-induced bronchial contraction in both air- and ozone-exposed C57/BL6 mice, but not in air- or ozone-exposed MKP-1(-/-) mice. ACh-stimulated p38 MAPK and heat shock protein (HSP)27 phosphorylation, as measured by Western blotting, and this effect was suppressed by SB239063 in C57/BL6 and MKP-1(-/-) mice, but not by dexamethasone in either air- or ozone-exposed MKP-1(-/-) mice. p38 MAPK plays a role in maximal ACh-induced isometric contractile responses and increased contractility induced by ozone. Dexamethasone inhibits ACh-induced ASM contraction through phosphorylation of p38 MAPK and HSP27.

Inhibitory effect of anticholinergics on the contraction of isolated caprine urinary bladder detrusor muscle

This study investigated whether four anticholinergics which are not clinically used for relaxing the urinary bladder detrusor muscle inhibit the contraction of isolated caprine (goat) detrusor muscle: cyclopentolate (100 nm), homatropine (5 microm), ipratropium (500 nm) and valethamate (1 microm). The effects of these anticholinergics were compared with tolterodine (3 microm), an anticholinergic clinically used for relaxing the detrusor muscle. The inhibitory effect of each of these five anticholinergics was studied on six strips of caprine detrusor muscle made to contract with 100 mum acetylcholine (ACh) by determining the percent inhibition of height of contraction and the area under the contractile curve (AUC). It was found that all five anticholinergics inhibited the ACh-induced contraction of the caprine detrusor and that this inhibition was reversed by raising the concentration of ACh. Hence, these four anticholinergics, like tolterodine, may be useful in managing clinical conditions that require relaxation of the detrusor muscle.

Phosphoinositide 3-Kinase γ Regulates Airway Smooth Muscle Contraction by Modulating Calcium Oscillations

Phosphoinositide 3-kinase gamma (PI3Kgamma) has been implicated in the pathogenesis of asthma, but its mechanism has been considered indirect, through release of inflammatory cell mediators. Because airway smooth muscle (ASM) contractile hyper-responsiveness plays a critical role in asthma, the aim of the present study was to determine whether PI3Kgamma can directly regulate contractility of ASM. Immunohistochemistry staining indicated expression of PI3Kgamma protein in ASM cells of mouse trachea and lung, which was confirmed by Western blot analysis in isolated mouse tracheal ASM cells. PI3Kgamma inhibitor II inhibited acetylcholine (ACh)-stimulated airway contraction of cultured precision-cut mouse lung slices in a dose-dependent manner with 75% inhibition at 10 muM. In contrast, inhibitors of PI3Kalpha, PI3Kbeta, or PI3Kdelta, at concentrations 40-fold higher than their reported IC(50) values for their primary targets, had no effect. It is noteworthy that airways in lung slices pretreated with PI3Kgamma inhibitor II still exhibited an ACh-induced initial contraction, but the sustained contraction was significantly reduced. Furthermore, the PI3Kgamma-selective inhibitor had a small inhibitory effect on the ACh-stimulated initial Ca(2+) transient in ASM cells of mouse lung slices or isolated mouse ASM cells but significantly attenuated the sustained Ca(2+) oscillations that are critical for sustained airway contraction. This report is the first to show that PI3Kgamma directly controls contractility of airways through regulation of Ca(2+) oscillations in ASM cells. Thus, in addition to effects on airway inflammation, PI3Kgamma inhibitors may also exert direct effects on the airway contraction that contribute to pathologic airway hyper-responsiveness.

Endothelium-dependent contraction induced by acetylcholine in the chicken ductus arteriosus involves cyclooxygenase-1 activation and TP receptor stimulation

In numerous vascular beds, acetylcholine (ACh) evokes the simultaneous release of endothelium-derived relaxing and contracting factors (EDRF and EDCF, respectively). We aimed to determine whether ACh evokes the release of an EDCF in the chicken ductus arteriosus (DA) and to identify its nature. Isolated rings DA from 19-d chicken embryos (total incubation: 21-d) were studied in a wire myograph. Low concentrations of ACh (30 nM–1 µM) elicited a relaxation, which was followed by a contraction at higher concentrations (3 µM–0.1 mM). Both relaxation and contraction were abolished by removal of endothelium and were sensitive to the antimuscarinic agents atropine and 4-DAMP (M 3-receptor antagonist). ACh-induced contraction was impaired in the presence of the non-selective inhibitor of cyclooxygenase (COX) indomethacin, the selective COX-1 inhibitor valeryl salicylate, and the thromboxane (TX)/prostaglandin (PG) H 2 (TP) receptor blocker SQ-29458, whereas the response was not affected by the selective COX-2 inhibitor nimesulide, the TX synthase inhibitor furegrelate, the H 2O 2 scavenger PEG-catalase, the nitric oxide synthase inhibitor L-NAME, or the soluble guanylate cyclase inhibitor ODQ. Enzyme immunoassay determined that, under basal conditions, the chicken DA produced PGE 2, PGF 2α and TXB 2 (stable metabolite of TXA 2). Prostanoid production was inhibited by indomethacin but was not significantly affected by ACh. We conclude that in the chicken DA, stimulation of muscarinic receptors by ACh induces an endothelium-dependent relaxation followed by an endothelium-dependent contraction. The contraction involves COX-1 activation and TP receptor stimulation.

Mechanisms underlying the losartan treatment-induced improvement in the endothelial dysfunction seen in mesenteric arteries from type 2 diabetic rats

It is well known that type 2 diabetes mellitus is frequently associated with vascular dysfunction and an elevated systemic blood pressure, yet the underlying mechanisms are not completely understood. We previously reported that in mesenteric arteries from established type 2 diabetic Otsuka Long-Evans Tokushima fatty (OLETF) rats, which exhibit endothelial dysfunction, there is an imbalance between endothelium-derived vasodilators [namely, nitric oxide (NO) and hyperpolarizing factor (EDHF)] and vasoconstrictors [contracting factors (EDCFs) such as cyclooxygenase (COX)-derived prostanoids]. Here, we investigated whether the angiotensin II receptor antagonist losartan might improve endothelial dysfunction in OLETF rats at the established stage of diabetes. In mesenteric arteries isolated from OLETF rats [vs. those from age-matched control Long-Evans Tokushima Otsuka (LETO) rats]: (1) the acetylcholine (ACh)-induced relaxation was impaired, (2) the NO- and EDHF-mediated relaxations were reduced, (3) the ACh-induced EDCF-mediated contraction and the production of prostanoids were increased, and (4) superoxide generation was increased. After such OLETF rats had received losartan (25 mg/kg/day p.o. for 4 weeks), their isolated mesenteric arteries exhibited: (1) improvements in ACh-induced NO- and EDHF-mediated relaxations, (2) reduced EDCF- and arachidonic acid-induced contractions, (3) suppressed production of prostanoids, (4) reduced PGE 2-mediated contraction, and (5) reduced superoxide generation. Within the timescale studied here, losartan did not change the protein expressions of endothelial NO synthase, COX1, or COX2 in mesenteric arteries from either OLETF or LETO rats. Losartan thus normalizes vascular dysfunction in this type 2 diabetic model, and the above effects may contribute to the reduction of adverse cardiovascular events seen in diabetic patients treated with angiotensin II receptor blockers.

The essential oil of Eucalyptus tereticornis, and its constituents α- and β-pinene, potentiate acetylcholine-induced contractions in isolated rat trachea

The effects of the essential oil of Eucalyptus tereticornis (EOET), especially the effects of its constituents α- and β-pinene, were studied on rat trachea in vitro. In tracheal rings, EOET, α- or β-pinene potentiated the contractions induced by acetylcholine (ACh). Contractions induced by K + (60 mM) were also potentiated by α- and β-pinene, but were reduced by EOET. Our findings show that EOET has myorelaxant effects on rat airways, but potentiates ACh-induced contractions. Monoterpenes α- and β-pinene are involved in its potentiating actions, but are not responsible for its myorelaxant effects. A putative inhibition of the acetylcholinesterase enzyme is involved.

Adenosine A2A agonist and A2B antagonist mediate an inhibition of inflammation-induced contractile disturbance of a rat gastrointestinal preparation

Adenosine can show anti-inflammatory as well as pro-inflammatory activities. The contribution of the specific adenosine receptor subtypes in various cells, tissues and organs is complex. In this study, we examined the effect of the adenosine A(2A) receptor agonist CGS 21680 and the A(2B)R antagonist PSB-1115 on acute inflammation induced experimentally by 2,4,6-trinitrobenzenesulfonic acid (TNBS) on rat ileum/jejunum preparations. Pre-incubation of the ileum/jejunum segments with TNBS for 30 min resulted in a concentration-dependent inhibition of acetylcholine (ACh)-induced contractions. Pharmacological activation of the A(2A)R with CGS 21680 (0.1-10 microM) pre-incubated simultaneously with TNBS (10 mM) prevented concentration-dependently the TNBS-induced inhibition of the ACh contractions. Stimulation of A(2B)R with the selective agonist BAY 60-6583 (10 microM) did neither result in an increase nor in a further decrease of ACh-induced contractions compared to the TNBS-induced inhibition. The simultaneous pre-incubation of the ileum/jejunum segments with TNBS (10 mM) and the selective A(2B)R antagonist PSB-1115 (100 microM) inhibited the contraction-decreasing effect of TNBS. The effects of the A(2A)R agonist and the A(2B)R antagonist were in the same range as the effect induced by 1 microM methotrexate. The combination of the A(2A)R agonist CGS 21680 and the A(2B)R antagonist PSB-1115 at subthreshold concentrations of both agents found a significant amelioration of the TNBS-diminished contractility. Our results demonstrate that the activation of A(2A) receptors or the blockade of the A(2B) receptors can prevent the inflammation-induced disturbance of the ACh-induced contraction in TNBS pre-treated small intestinal preparations. The combination of both may be useful for the treatment of inflammatory bowel diseases.