Carbachol-evoked Contractions Research Articles

M2 Muscarinic Receptor-Dependent Contractions of Airway Smooth Muscle are Inhibited by Activation of β-Adrenoceptors.

Beta-adrenoceptor (β-AR) agonists inhibit cholinergic contractions of airway smooth muscle (ASM), but the underlying mechanisms are unclear. ASM cells express M3 and M2 muscarinic receptors, but the bronchoconstrictor effects of acetylcholine are believed to result from activation of M3Rs, while the role of the M2Rs is confined to offsetting β-AR-dependent relaxations. However, a profound M2R-mediated hypersensitization of M3R-dependent contractions of ASM was recently reported, indicating an important role for M2Rs in cholinergic contractions of ASM. Here, we investigated if M2R-dependent contractions of murine bronchial rings were inhibited by activation of β-ARs. M2R-dependent contractions were apparent at low frequency (2Hz)electric field stimulation (EFS) and short (10s) stimulus intervals.The β1-AR agonist, denopamine inhibited EFS-evoked contractions of ASM induced by reduction in stimulus interval from 100 to 10 sand was more effective at inhibiting contractions evoked by EFS at 2than 20 Hz.Denopamine also abolished carbachol-evoked contractions that were resistant to the M3R antagonist 4-DAMP, similar to the effects of the M2R antagonists, methoctramine and AFDX-116. The inhibitory effects of denopamine on EFS-evoked contractions of ASM were smaller in preparations taken from M2R -/- mice, compared to wild-type (WT) controls. In contrast, inhibitory effects of the β3-AR agonist, BRL37344, on EFS-evoked contractions of detrusor strips taken from M2R -/- mice were greater than WT controls. These data suggest that M2R-dependent contractions of ASM were inhibited by activation of β1-ARs and that genetic ablation of M2Rs decreased the efficacy of β-AR agonists on cholinergic contractions.

Trimetazidine attenuates cyclophosphamide-induced cystitis by inhibiting TLR4-mediated NFκB signaling in mice

AimCyclophosphamide (CP)-induced cystitis is a challenging clinical problem involving inflammation and dysfunction of bladder. Trimetazidine (TMZ) is an anti-anginal drug with anti-oxidant and anti-inflammatory properties. We aimed to investigate the protective effects of TMZ in CP-induced cystitis via inhibiting TLR4/NFκB signaling. Main methodsBalb/c mice were administrated TMZ (10 or 20 mg/kg/day) intraperitoneally (i.p.) for 5 consecutive days before CP. On day 6, cystitis was induced by a single dose of CP (300 mg/kg, i.p.). Mesna (2-mercaptoethane sulfonate sodium; 30 mg/kg, i.p.) was administered 20 min before and at 4 and 8 h after the CP injection. After 24 h of cystitis induction, the bladders were removed for histopathological evaluation, contractility studies, biochemical analysis and western blotting. MTT assay was performed in a cancer cell line (MDA-MB-231) to evaluate the effect of TMZ on the cytotoxicity of CP. Key findingsCP-induced severe cystitis was confirmed by histological disturbances and the decrease in carbachol-evoked contractions of detrusor strips, which was partially improved by TMZ (20 mg/kg/day). SOD activity and GSH content were decreased whereas TNF-α and IL-1β levels were increased in the bladders of CP-treated mice, which were restored by TMZ or mesna. TMZ reduced the CP-induced increase in the protein expressions of caspase-3, TLR4 and phosphorylated-NFκB in bladder tissues. TMZ alone decreased the cell viability and TMZ also enhanced the cytotoxicity of CP. SignificanceOur study provides the first preclinical evidence that TMZ attenuates CP-induced urotoxicity by enhancing anti-oxidant capacity and suppressing inflammation possibly via downregulating TLR4-mediated NFκB signaling while augmenting the cytotoxicity of CP.

Detrusor underactivity causes neurogenic voiding dysfunction in a rat bilateral accessory nerve-injury model

We aimed to investigate detrusor function in a previously developed rat neurogenic voiding dysfunction model that we have developed previously. We performed sham or bilateral accessory nerve injury (BACNI) surgeries on ten-week-old male Wistar/ST rats. One week after surgery, we evaluated detrusor contractility in the bladder using isometric tension and mRNA expression assays. Cholinergic contraction was attenuated in the injury model, whereas carbachol-evoked contraction was enhanced, and mRNA expression of the cholinergic receptor increased. These findings suggest that there was a reduction in neurotransmitter release causing detrusor underactivity.

Short chain fatty acids and colon motility in a mouse model of irritable bowel syndrome

BackgroundIrritable bowel syndrome (IBS) is defined as a multifactorial disorder associated with visceral hypersensitivity, altered gut motility and dysfunction of the brain-gut axis. Gut microbiota and its metabolites are proposed as possible etiological factors of IBS. Short chain fatty acids (SCFAs) induce both inhibitory and stimulatory action on colon motility, however, their effects on the IBS model were not investigated. The aim of our study was to investigate the level of SFCAs in feces and their effects on colon motility in a mouse model of IBS.MethodsIBS model was induced in mice by intracolonic infusion of 1% acetic acid during the early postnatal period. Mice colon hypersensitivity was assessed by the threshold of the abdominal withdrawal reflex in response to colorectal distention. Colon contractility was studied using proximal colon specimens in isometric conditions. Transit rates were assessed by the pellet propulsion in the isolated colon. Concentrations of SCFAs in feces were measured using gas–liquid chromatography.ResultsThe concentration of SCFAs in feces of IBS model mice was higher compared to the control group. Visceral sensitivity to colorectal distension and colonic transit rate were increased indicating IBS with predominant diarrhea. The frequency and amplitude of spontaneous contractions of proximal colon segments from IBS mice were higher, but carbachol induced contractions were lower compared to control. During acute application of SCFAs (sodium propionate, sodium acetate or butyric acid) dose-dependently (0.5–30 mM) decreased tonic tension, frequency and amplitude of spontaneous and carbachol-evoked contractions. In the mouse IBS group the inhibitory effects SCFAs on spontaneous and carbachol-evoked contractions were less pronounced. At the same time intraluminal administration of butyrate (5 mM) increased the transit rate in the colon of both groups, but its stimulatory effect was more pronounced in mouse IBS model group.ConclusionOur data indicate that the increased transit rate in the mouse IBS model group is associated with a disbalance of activating and inhibiting action of SCFAs due to chronically elevated SCFA levels, which may impact the pathogenesis of IBS with predominant diarrhea syndrome.

Relaxant effect of Lippia origanoides essential oil in guinea-pig trachea smooth muscle involves potassium channels and soluble guanylyl cyclase

Ethnopharmacological relevanceLippia origanoides H.B.K. is an aromatic species used in folk medicine to treat respiratory diseases, including asthma. Aim of the studyThe aim of this work was to evaluate the relaxing potential and mechanism of action of the L. origanoides (LOO) essential oil in isolated guinea-pig trachea. Materials and methodsLeaves from L. origanoides were collected at experimental fields under organic cultivation, at the Forest Garden of Universidade Estadual de Feira de Santana. Essential oil was extracted by hydrodistillation, analyzed by GC/FID and GC/MS and the volatile constituents were identified. Spasmolytic activity and relaxant mechanism of LOO were assayed in isolated guinea-pig trachea contracted with histamine, carbachol or hyperpolarizing KCl. ResultsChemical analysis revealed the presence of carvacrol (53.89%) as major constituent. LOO relaxed isolated guinea-pig trachea pre-contracted with KCl 60 mM [EC50 = 30.02 μg/mL], histamine 1 µM [EC50 = 9.28 μg/mL] or carbachol 1 µM [EC50 = 51.80 μg/mL]. The pre-incubation of glibenclamide, CsCl, propranolol, indomethacin, hexamethonium, aminophylline or L-NAME in histamine-induced contractions did not alter significantly the relaxant effect of LOO. However, the presence of 4-aminopyridine, tetraethylammonium or methylene blue reduced LOO effect, while the presence of dexamethasone or atropine potentialized the LOO relaxant effect. LOO pre-incubation inhibited carbachol-evoked contractions, with this effect potentialized in the presence of sodium nitroprusside and blocked in the presence of ODQ. ConclusionsThe relaxant mechanism of LOO on the tracheal smooth muscle possibly involves stimulating of soluble guanylyl cyclase with consequent activation of the voltage-gated and Ca2+-activated K+ channels.

Mucosal modulation of contractility in bladder strips from normal and overactive rat models and the effect of botulinum toxin A on overactive bladder strips.

To investigate the local, regulatory role of the mucosa on bladder strip contractility from normal and overactive bladders and to examine the effect of botulinum toxin A (BoNT-A). Bladder strips from spontaneously hyperactive rat (SHR) or normal rats (Sprague Dawley, SD) were dissected for myography as intact or mucosa-free preparations. Spontaneous, neurogenic and agonist-evoked contractions were investigated. SHR strips were incubated in BoNT-A (3 h) to assess effects on contractility. Spontaneous contraction amplitude, force-integral or frequency were not significantly different in SHR mucosa-free strips compared with intacts. In contrast, spontaneous contraction amplitude and force-integral were smaller in SD mucosa-free strips than in intacts; frequency was not affected by the mucosa. Frequency of spontaneous contractions in SHR strips was significantly greater than in SD strips. Neurogenic contractions in mucosa-free SHR and SD strips at higher frequencies were smaller than in intact strips. The mucosa did not affect carbachol-evoked contractions in intact versus mucosa-free strips from SHR or SD bladders. BoNT-A reduced spontaneous contractions in SHR intact strips; this trend was also observed in mucosa-free strips but was not significant. Neurogenic and carbachol-evoked contractions were reduced by BoNT-A in mucosa-free but not intact strips. Depolarisation-induced contractions were smaller in BoNT-A-treated mucosa-free strips. The mucosal layer positively modulates spontaneous contractions in strips from normal SD but not overactive SHR bladder strips. The novel finding of BoNT-A reduction of contractions in SHR mucosa-free strips indicates actions on the detrusor, independent of its classical action on neuronal SNARE complexes.

The Involvement of Ca(2+) Signal Pathways in Distal Colonic Myocytes in a Rat Model of Dextran Sulfate Sodium-induced Colitis.

Background:Disrupted Ca2+ homeostasis contributes to the development of colonic dysmotility in ulcerative colitis (UC), but the underlying mechanisms are unknown. This study aimed to examine the alteration of colonic smooth muscle (SM) Ca2+ signaling and Ca2+ handling proteins in a rat model of dextran sulfate sodium (DSS)-induced UC.Methods:Male Sprague-Dawley rats were randomly divided into control (n = 18) and DSS (n = 17) groups. Acute colitis was induced by 5% DSS in the drinking water for 7 days. Contractility of colonic SM strips (controls, n = 8 and DSS, n = 7) was measured in an organ bath. Cytosolic resting Ca2+ levels (n = 3 in each group) and Ca2+ transients (n = 3 in each group) were measured in single colonic SM cells. Ca2+ handling protein expression was determined by Western blotting (n = 4 in each group). Differences between control and DSS groups were analyzed by a two-sample independent t-test.Results:Average tension and amplitude of spontaneous contractions of colonic muscle strips were significantly enhanced in DSS-treated rats compared with controls (1.25 ± 0.08 g vs. 0.96 ± 0.05 g, P = 0.007; and 2.67 ± 0.62 g vs. 0.52 ± 0.10 g, P = 0.013). Average tensions of carbachol-evoked contractions were much weaker in the DSS group (1.08 ± 0.10 g vs. 1.80 ± 0.19 g, P = 0.006). Spontaneous Ca2+ transients were observed in more SM cells from DSS-treated rats (15/30 cells) than from controls (5/36 cells). Peak caffeine-induced intracellular Ca2+ release was lower in SM cells of DSS-treated rats than controls (0.413 ± 0.046 vs. 0.548 ± 0.041, P = 0.033). Finally, several Ca2+ handling proteins in colonic SM were altered by DSS treatment, including sarcoplasmic reticulum calcium-transporting ATPase 2a downregulation and phospholamban and inositol 1,4,5-trisphosphate receptor 1 upregulation.Conclusions:Impaired intracellular Ca2+ signaling of colonic SM, caused by alteration of Ca2+ handing proteins, contribute to colonic dysmotility in DSS-induced UC.

Izalpinin from fruits of Alpinia oxyphylla with antagonistic activity against the rat bladder contractility.

Alpinia oxyphylla (Zingiberaceae), an herbaceous perennial plant, its capsular fruit is commonly used in traditional Chinese medicine for the treatment of different urinary incontinence symptoms including frequency, urgency and nocturia. These symptoms are similar to the overactive bladder syndrome. In our lab, we found that the 95% ethanol extract of the capsular fruits exhibited significant anti-muscarinic activity. Some constituents in capsular fruits including flavonoids (e.g., izalpinin and tectochrysin), diarylheptanoids (e.g., yakuchinone A and yakuchinone B) and sesquiterpenes (e.g., nootkatone), are regarded as representative chemicals with putative pharmacological activities. This study aimed to evaluate the in vitro antagonistic actions of izalpinin on carbachol-induced contraction of the rat detrusor muscle. In vitro inhibition of rat detrusor contractile response to carbachol was used to study the functional activity of izalpinin. The isolated detrusor strips of rats were mounted in organ baths containing oxygenated Krebs' solution. The cumulative consecutive concentration-response curves to carbachol-evoked contractions in strips of rat bladder were obtained. Carbachol induced concentration-dependent contractions of isolated rat bladder detrusor strips. The vehicle DMSO had no impact on the contraction response. The contraction effects were concentration-dependently antagonized by izalpinin, with a mean EC50 value of 0.35 µM. The corresponding cumulative agonist concentration-response curves shifted right-ward. Izalpinin exhibits inhibitory role of muscarinic receptor-related detrusor contractile activity, and it may be a promising lead compound to treat overactive bladder.

Role of cyclooxygenase isoforms in the altered excitatory motor pathways of human colon with diverticular disease

The COX isoforms (COX-1, COX-2) regulate human gut motility, although their role under pathological conditions remains unclear. This study examines the effects of COX inhibitors on excitatory motility in colonic tissue from patients with diverticular disease (DD). Longitudinal muscle preparations, from patients with DD or uncomplicated cancer (controls), were set up in organ baths and connected to isotonic transducers. Indomethacin (COX-1/COX-2 inhibitor), SC-560 (COX-1 inhibitor) or DFU (COX-2 inhibitor) were assayed on electrically evoked, neurogenic, cholinergic and tachykininergic contractions, or carbachol- and substance P (SP)-induced myogenic contractions. Distribution and expression of COX isoforms in the neuromuscular compartment were assessed by RT-PCR, Western blot and immunohistochemical analysis. In control preparations, neurogenic cholinergic contractions were enhanced by COX inhibitors, whereas tachykininergic responses were blunted. Carbachol-evoked contractions were increased by indomethacin or SC-560, but not DFU, whereas all inhibitors reduced SP-induced motor responses. In preparations from DD patients, COX inhibitors did not affect electrically evoked cholinergic contractions. Both indomethacin and DFU, but not SC-560, decreased tachykininergic responses. COX inhibitors did not modify carbachol-evoked motor responses, whereas they counteracted SP-induced contractions. COX-1 expression was decreased in myenteric neurons, whereas COX-2 was enhanced in glial cells and smooth muscle. In control colon, COX-1 and COX-2 down-regulate cholinergic motility, whereas both isoforms enhance tachykininergic motor activity. In the presence of DD, there is a loss of modulation by both COX isoforms on the cholinergic system, whereas COX-2 displays an enhanced facilitatory control on tachykininergic contractile activity.

Cannabinoid receptors 1 and 2 are associated with bladder dysfunction in an experimental diabetic rat model

To investigate diabetes-associated changes in urinary bladder expression of cannabinoid receptors 1 and 2 (CB1 and CB2) and the functional role of CB agonists and antagonists in mediating phasic contractions of isolated bladder strips using a streptozotocin-induced diabetic rat model. The bladder and dorsal root ganglion (DRG) were removed from diabetic rats and age-matched controls 8-10 weeks after diabetes induction. Expression of CB1 and CB2 mRNA was studied using quantitative real-time PCR and protein levels were determined by Western blot analysis. The effect of increasing concentrations (0.1-100 μM) of the mixed CB1/CB2 agonist R(+)-WIN 55,212-2 (WIN), selective CB1 antagonist (AM251) and selective CB2 antagonist (AM630) on carbachol-evoked contraction of bladder strips from control and diabetic rats was investigated. WIN-induced alterations of bladder strip contraction were then studied after pre-incubation with AM251 and AM630. Diabetes induced decreased CB1 protein and mRNA expression in both the bladder and DRG (P < 0.05), while decreased CB2 expression was observed in the bladder (P < 0.05). WIN decreased the amplitude, but not frequency, of carbachol-induced phasic contractions of bladder strips in a concentration-dependent manner and this effect was diminished in the diabetic state. AM630 and AM251 had no effect on isolated detrusor muscle function. Moreover, pre-incubation with AM251 partially counteracted the effect of WIN on detrusor muscle contraction. The results indicate that CB1 and CB2 are responsible for the pathogenesis of bladder dysfunction in diabetes mellitus and represent a viable target for pharmacological treatment of bladder cystopathy.

Su2046 Delayed Gastrointestinal Transit in OB/OB Mice Correlates With Reduced Intestinal TLR4 Signaling and Loss of Nitrergic Neurons

100 μM). L-732,138 (NK1 receptor antagonist, 10 μM) or atropine (1 μM) were used to record contractions driven primarily by acetylcholine or tachykinins, respectively. In addition, contractions elicited by exogenous substance P (SP, 10 μM) or carbachol (10 μM), in the presence of tetrodotoxin (1 μM), were assessed. Electrically evoked contractile responses (5 Hz) in the presence L-NAME were also recorded and expressed as % of contractions in the absence of L-NAME. Each value represents the mean ± S.E.M obtained from 6 experiments. Results. Electrically evoked cholinergic contractions were decreased in rats sacrificed at day 28 and 56 after 6-OHDA injection (77.2±6.3 and 53.8±0.1, respectively), in comparison with controls (99±6.51). Conversely, carbachol-evoked contractions were enhanced in rats with PD both at day 28 and 56 (81.5±3.4 and 98.2±3.8, respectively), as compared to controls (52.2±5.6). Electrically evoked tachykininergic contractions were enhanced both at day 28 (85.6±5.2) and 56 (67.3±3.3), in comparison with controls (43.6±6.5). Likewise, contractions elicited by exogenous SP in colonic preparations from PD rats were also increased both at day 28 (244±11.9) and 56 (188.2±13.9), as compared to controls (79.6±6.3). In the presence of L-NAME, electrically evoked contractions in controls were enhanced (+42.3±11.9%), while they were decreased in rats at day 28 and 56 after 6-OHDA injection (+29.1±4.4% and +15±5.3%, respectively). Conclusion. Experimental PD, elicited by nigrostriatal dopaminergic degeneration, is associated with changes in neurotransmitter pathways driving colonic excitatory and inhibitory motor functions: an impairment of nitrergic and cholinergic transmission occurs in concomitance with an enhancement of tachykininergic control. Such a shift takes place along with an up-regulation of the contractile responses mediated bymuscarinic receptors on smoothmuscle, whichmight be compensatory in nature.

Guinea-pig ileum as ex vivo model useful to characterize ligands displaying Imidazoline I2 and Adrenergic alpha2 mixed activity: a preliminary study

The lack of an effective analgesic treatment makes pain a clinical challenge and the need of a novel approach to identify new agents is urgent. In this scenario I2-ligands can be considered an alternative strategy in pain therapy. The development of an &lt;em&gt;ex vivo&lt;/em&gt; model useful for the evaluation of functional activities at both a2 and I2-IBs (imidazoline binding sites) is an important task in pharmacological sciences since several I2 ligands display activity also towards a receptors. The present study aims to develop an &lt;em&gt;ex vivo&lt;/em&gt; model for estimating the activity of I2-IBs ligands in a biological sample where a1 and a2 adrenergic receptors are present. For this purpose the imidalzoline endogenous ligand, harmane, reference compounds, 2BFI and BU224, and imidazoline derivatives 1-3 have been selected taking into account their in vitro activity towards IBs and adrenergic receptors. All compounds have been tested &lt;em&gt;ex vivo&lt;/em&gt; in guinea pig-ileum where a2A-ARs are prejunctionally and I2-IBS postjunctionally localized. Adrenergic component has been identified by the studying the interference of compounds on the electrically-evoked contraction while I2-IBs activity by testing the ability of compounds to inhibit the carbachol-evoked contractions in the presence of prazosin to mask the a1 adrenoceptors. Compounds 1 and 2 were found I2-IBs antago nists (pIC50=4.2 and 4.0, respectively) whereas compound 3 was I2-IBs agonist (EC50=0.38 mM); All ligands were a2 adrenergic agonists. This paper suggests guinea-pig ileum as the first &lt;em&gt;ex vivo&lt;/em&gt; approach for establishing both the intrinsic activity of I2-IBs ligands and the physiological correlation between IBs and adrenergic system.

Kcnq Channels in Airway Smooth Muscle

The role of KCNQ (Kv7) channels is well established in neurons, where they play dominant roles in control of resting membrane potentials and cell excitability. Recent studies have revealed expression of KCNQ channels in different types of smooth muscle. Here we focused on airway smooth muscle (ASM), whose membrane potential is primarily, determined by BK-type K+ channels. RT-PCR and immunostaining suggest that KCNQ 1, 4 and 5 are the predominant subtypes in rodent airway. To investigate the contribution of KCNQ channels in cholinergic-induced ASM contraction, we patch-clamped freshly isolated ASM cells and isolated KCNQ current as the non-inactivating component at the end of 2s depolarizations, with BK channels blocked with 1μM paxilline. The KCNQ current was enhanced by the KCNQ channel opener, flupirtine (10μM), and abolished by XE991 (10μM). Although XE991 depolarized the resting membrane potential, it did not affect that of ASM cells pre-treated with carbachol. Similarly, XE991 did not effect carbachol-evoked contractions, whereas flupirtine induced a significant relaxation. The flupirtine effect is likely via opposing voltage-dependent Ca2+ influx, since no effect of flupertine occurred in a low K+ (1mM) bathing solution. Pre-treatment with the muscarinic M3-receptor antagonist, fumarate (5nM), to prevent Gq/11-mediated PIP2 hydrolysis did not reveal an effect of XE991 on contractions, suggesting the mechanism of KCNQ inhibition in ASM is not due to PIP2 depletion. Interestingly, an XE991-dependent increase in contractility was revealed in mice with the β1 BK channel subunit knocked out, an effect enhanced by the M3 receptor antagonist. Thus, KCNQ channels may normally play a secondary role to BK channels in control of cholinergic evoked contractions in ASM; however, given that alteration in BK β1-subunit expression is known to occur in asthma, KCNQ channels in ASM cells may be potential targets for therapeutic intervention in respiratory disease.

Muscarinic receptor subtypes involved in urothelium-derived relaxatory effects in the inflamed rat urinary bladder

Functional studies have shown altered cholinergic mechanisms in the inflamed bladder, which partly depend on muscarinic receptor-induced release of nitric oxide (NO). The current study aimed to characterize which muscarinic receptor subtypes that are involved in the regulation of the nitrergic effects in the bladder cholinergic response during cystitis. For this purpose, in vitro examinations of carbachol-evoked contractions of inflamed and normal bladder preparations were performed. The effects of antagonists with different selectivity for the receptor subtypes were assessed on intact and urothelium-denuded bladder preparations. In preparations from cyclophosphamide (CYP; in order to induce cystitis) pre-treated rats, the response to carbachol was about 75% of that of normal preparations. Removal of the urothelium or administration of a nitric oxide synthase inhibitor re-established the responses in the inflamed preparations. Administration of 4-diphenylacetoxy-N-methylpiperidine (4-DAMP) inhibited the carbachol-induced contractile responses of preparations from CYP pre-treated rats less potently than controls. Pirenzepine and p-fluoro-hexahydro-sila-diphenidol (pFHHSiD) affected the carbachol-induced contractile responses to similar extents in preparations of CYP pre-treated and control rats. However, the Schild slopes for the three antagonists were all significantly different from unity in the preparations from CYP pre-treated rats. Again, L-NNA or removal of the urothelium eliminated any difference compared to normal preparations. This study confirms that muscarinic receptor stimulation in the inflamed rat urinary bladder induces urothelial release of NO, which counteracts detrusor contraction.

Inhibitory effects of indicaxanthin on mouse ileal contractility: Analysis of the mechanism of action

Recently, we have showed that indicaxanthin, the yellow betalain pigment abundant in the fruit of Opuntia ficus indica, has remarkable spasmolytic effects on the intestinal contractility in vitro. Thus, the purpose of the present study was to investigate the mechanism of action underlying the observed response. We used organ bath technique to record the mechanical activity of the mouse ileum longitudinal muscle and ELISA to measure the levels of cAMP. Indicaxanthin induced inhibitory effects on spontaneous mechanical activity, which were unaffected by indomethacin, a non-selective inhibitor of cycloxygenase; 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, a selective inhibitor of nitric oxide-dependent guanylyl cyclase; 2′5′dideoxyadenosine, an adenylyl cyclase inhibitor; and zaprinast, a selective inhibitor of the cGMP phosphodiesterase isoenzyme. Indicaxanthin effects were reduced significantly in the presence of 3-isobutyl-1-methylxanthine (IBMX), a non selective inhibitor of phosphodiesterases (PDEs). Indicaxanthin and IBMX significantly reduced the carbachol-evoked contractions and the joint application of both drugs did not produce any additive effect. Indicaxanthin and IBMX increased the inhibitory effects of forskolin, an adenylyl cyclase activator, and the joint application of both drugs did not produce any additive effect. Indicaxanthin, contrarily to IBMX, did not affect the inhibitory action of sodium nitroprusside, a soluble guanylyl cyclase activator. Indicaxanthin increased both basal and forskolin-induced cAMP content of mouse ileal muscle. The present data show that indicaxanthin reduces the contractility of ileal longitudinal muscle by inhibition of PDEs and increase of cAMP concentration and raise the possibility of using indicaxanthin in the treatment of motility disorders, such as abdominal cramps.

Involvement of Secondary Intracellular Messengers in the Mechanisms of Purinergic Inhibition of Intestinal Smooth Muscles

We studied the role of adenylate cyclase and phospholipase C in the control of ATP-induced relaxation of carbachol-evoked contraction of smooth muscles of the guinea-pig taenia coli. We showed that ATP-induced relaxation of carbachol-caused contraction is completely realized under control conditions via activation of inositol trisphosphate-sensitive (InsP3) receptors of the sarcoplasmic reticulum of smooth muscle cells (SMCs). In the case where phospholipase C was blocked, the relaxing action of ATP on smooth muscles continues to be mediated mostly by activation of InsP3 receptors, but other mechanisms begin to participate in this process. Intracellular processes are also involved in ATP-induced relaxation where the signal is transferred from purine receptors via activation of phospholipase C under conditions of parallel activation of adenylate cyclase by forskolin; these processes also include activation of InsP3 receptors of the sarcoplasmic reticulum of SMCs and some other events. After U73122-induced blocking of phospholipase C and forskolin-induced activation of adenylate cyclase, ATP-caused relaxation can completely be removed by an inhibitor of InsP3 receptors, 2-APB. This indicates that, under the above conditions, such relaxation is realized exclusively via InsP3 receptors of the sarcoplasmic reticulum of SMCs. At the same time, ATP-induced relaxation caused by activation of phospholipase C and inactivation of adenylate cyclase is also nearly completely realized with involvement of InsP3 receptors of the sarcoplasmic reticulum. However, removing the activity of phospholipase C under conditions of blocking of adenylate cyclase and InsP3 receptors of the sarcoplasmic reticulum in SMCs leads to the recovery of ATP-induced relaxation with the participation of the other intracellular processes. Therefore, two intracellular messengers, phospholipase C and adenylate cyclase, are involved in purinergic inhibition of smooth muscles. Upon their action, multiple intracellular signal pathways are triggered. The level of their participation can be influenced by the initial functional state of intestinal SMCs. These changes are always directed toward the maintenance of normal functioning of the respective organs.

Differential recruitment of high affinity A 1 and A 2A adenosine receptors in the control of colonic neuromuscular function in experimental colitis

This study investigated the expression of A 1 and A 2A receptors in the rat colonic neuromuscular compartment, and characterized their roles in the control of motility during inflammation. Colitis was induced by 2,4-dinitrobenzenesulfonic acid. A 1, A 2A receptors, and ecto-5′-nucleotidase (CD73, adenosine producing enzyme) mRNA expression was examined by RT-PCR. The effects of DPCPX (A 1 receptor antagonist), CCPA (A 1 receptor agonist), 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol (A 2A receptor antagonist), 4-[2-[[6-amino-9-(N-ethyl-b- d-ribofuranuronamidosyl)-9H-purin-2-yl]amino]ethyl]benzenepropanoic acid hydrochloride (A 2A receptor agonist), AOPCP (CD73 inhibitor) were tested on electrically or carbachol-evoked contractions in colonic longitudinal muscle preparations. In normal colon, RT-PCR revealed the presence of A 1 receptors, A 2A receptors and CD73, and an increased expression of A 2A receptors and CD73 was detected in inflamed tissues. In normal colon, DPCPX or 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol enhanced electrically-induced contractions, while in inflamed preparations the effect of DPCPX no longer occurred. In normal colon, CCPA or 4-[2-[[6-amino-9-(N-ethyl-b- d-ribofuranuronamidosyl)-9H-purin-2-yl]amino]ethyl] benzenepropanoic acid hydrochloride decreased electrically-induced contractions. Under inflammation, 4-[2-[[6-amino-9-(N-ethyl-b- d-ribofuranuronamidosyl)-9H-purin-2-yl]amino]ethyl] benzenepropanoic acid hydrochloride reduced electrically evoked contractions with higher efficacy, while the inhibition by CCPA remained unchanged. A 1 and A 2A receptor ligands did not affect carbachol-induced contractions. AOPCP enhanced electrically-induced contractions and prevented the contractile effects of 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol, without interfering with DPCPX, both in normal and inflamed colons. These results indicate that, in normal colon, both A 1 and A 2A receptors contribute to the inhibitory control of motor functions at neuronal level. Under bowel inflammation, A 1 receptor loses its modulating actions, while the recruitment of A 2A receptor by CD73-dependent endogenous adenosine drives an enhanced inhibitory control of colonic neuromotility.

A1 and A2a receptors mediate inhibitory effects of adenosine on the motor activity of human colon

Experimental evidence in animal models suggests that adenosine is involved in the regulation of digestive functions. This study examines the influence of adenosine on the contractile activity of human colon. Reverse transcription-polymerase chain reaction revealed A(1) and A(2a) receptor expression in colonic neuromuscular layers. Circular muscle preparations were connected to isotonic transducers to determine the effects of 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; A(1) receptor antagonist), ZM 241385 (A(2a) receptor antagonist), CCPA (A(1) receptor agonist) and 2-[(p-2-carboxyethyl)-phenethylamino]-5'-N-ethyl-carboxamide-adenosine (CGS 21680; A(2a) receptor agonist) on motor responses evoked by electrical stimulation or carbachol. Electrically evoked contractions were enhanced by DPCPX and ZM 241385, and reduced by CCPA and CGS 21680. Similar effects were observed when colonic preparations were incubated with guanethidine (noradrenergic blocker), L-732,138, GR-159897 and SB-218795 (NK receptor antagonists). However, in the presence of guanethidine, NK receptor antagonists and N(omega)-propyl-L-arginine (NPA; neuronal nitric oxide synthase inhibitor), the effects of DPCPX and CCPA were still evident, while those of ZM 241385 and CGS 21680 no longer occurred. Carbachol-induced contractions were unaffected by A(2a) receptor ligands, but they were enhanced or reduced by DPCPX and CCPA, respectively. When colonic preparations were incubated with guanethidine, NK antagonists and atropine, electrically induced relaxations were partly reduced by ZM 241385 or NPA, but unaffected by DPCPX. Dipyridamole or application of exogenous adenosine reduced electrically and carbachol-evoked contractions, whereas adenosine deaminase enhanced such motor responses. In conclusion, adenosine exerts an inhibitory control on human colonic motility. A(1) receptors mediate direct modulating actions on smooth muscle, whereas A(2a) receptors operate through inhibitory nitrergic nerve pathways.

Calcium Sensitization in Human Esophageal Muscle: Role for RhoA Kinase in Maintenance of Lower Esophageal Sphincter Tone

A rise in intracellular-free calcium ([Ca(2+)](i)) concentration is important for initiating contraction of smooth muscles, and Ca(2+) sensitization involving RhoA kinase can sustain tension. We previously found that [Ca(2+)](i) was comparable in cells from the esophageal body (EB) and lower esophageal sphincter (LES) muscles, despite the fact that the LES maintains resting tone. We hypothesized that Ca(2+) sensitization contributes to contraction in human esophageal muscle. Tension and [Ca(2+)](i) were measured simultaneously in intact human EB and LES muscles using the ratiometric Ca(2+)-sensitive dye fura-2. Spontaneous oscillations in EB muscle tension were associated with transient elevations of [Ca(2+)](i). Carbachol caused a large increase in tension, compared with spontaneous oscillations, although the rise of [Ca(2+)](i) was similar, suggesting Ca(2+) sensitization. The RhoA-kinase blockers (R)-(+)-trans-4-(1-aminoethyl)-N-(4-pyridyl) cyclohexanecarboxamide dihydrochloride monohydrate (Y-27632) and 1-(5-isoquinolinesulfonyl)-homopiperazine hydrochloride (HA-1077) reduced carbachol- and nerve-evoked contraction of the EB, accompanied by smaller reduction in the rise of [Ca(2+)](i). Protein kinase C inhibitors reduced force to a lesser extent. RhoA-kinase blockers caused concentration-dependent reduction of tension in spontaneously contracted LES muscles. Moreover, RhoA-kinase blockers reduced intrinsic nerve-evoked and carbachol-evoked contraction. However, there was no effect on nerve- or nitric oxide-mediated relaxation of LES. Ca(2+) sensitization mediated by the RhoA-kinase pathway has an important role in contraction of human EB muscles and LES tonic contraction, a feature not previously recognized.

Using Caveolin-1 Knockout Mouse to Study Impaired Detrusor Contractility and Disrupted Muscarinic Activity in the Aging Bladder

Objectives The caveolin-1 knockout mouse has been proposed as an animal model to study impaired bladder contractility and detrusor overactivity. This study investigated the effects of aging on detrusor contraction in wild type and caveolin-1 knockout mice. Methods Young (3-month-old) and old (1-year-old) male caveolin-1 knockout mice and their age-matched male wild type littermates (controls) were used. Longitudinal bladder strips were stimulated electrically (20 Hz) and pharmacologically using 1 to 10 μM carbachol (a nonsubtype selective cholinergic receptor agonist), 10 μM α,β-methylene adenosine triphosphate (a purinergic receptor agonist), and 100 mM potassium (a depolarizing agent). Isometric bladder strip contractions were compared between the young wild type and knockout groups and between the old wild type and knockout groups. Results The bladder strips from the 1-year-old knockout mice exhibited a 40% to 42% decrease in electrical neural contractions and carbachol-evoked contractions compared with the 1-year-old wild type controls ( P <0.05). Even though the bladder strips from the 3-month-old knockout mice demonstrated a smaller decrease (29% to 32%) in electrical neural contractions and carbachol-evoked contractions compared with their age-matched controls, the trend did not reach statistical significance ( P >0.05). The postjunctional cholinergic pathway was specifically disrupted in caveolin-1 knockout animals because no difference was found in contractility between the knockout and wild type mice (young or old) when the bladders were stimulated by α,β-methylene adenosine triphosphate or potassium. The differences in cholinergic contractility between the knockout and wild type mice became significantly greater as the animals aged from 3 months old (young bladders) to 1 year old (aged bladders). Conclusions The male caveolin-1 knockout mouse provides a much-needed animal model for the study of impaired detrusor contractility in the aging bladder.