Carbachol-induced Contraction Research Articles

Involvement of the Tyr Kinase/JNK Pathway in Carbachol-induced Bronchial Smooth Muscle Contraction in the Rat

Tyrosine (Tyr) kinases and mitogen-activated protein kinases have been thought to participate in the contractile response in various smooth muscles. The aim of the current study was to investigate the involvement of the Tyr kinase pathway in the contraction of bronchial smooth muscle. Ring preparations of bronchi isolated from rats were suspended in an organ bath. Isometric contraction of circular smooth muscle was measured. Immunoblotting was used to examine the phosphorylation of c-Jun N-terminal kinasess (JNKs) in bronchial smooth muscle. To examine the role of mitogen-activated protein kinase(s) in bronchial smooth muscle contraction, the effects of MPAK inhibitors were investigated in this study. The contraction induced by carbachol (CCh) was significantly inhibited by pretreatment with selective Tyr kinase inhibitors (genistein and ST638, n = 6, respectively), and a JNK inhibitor (SP600125, n = 6). The contractions induced by high K depolarization (n = 4), orthovanadate (a potent Tyr phosphatase inhibitor) and sodium fluoride (a G protein activator; NaF) were also significantly inhibited by selective Tyr kinase inhibitors and a JNK inhibitor (n = 4, respectively). However, the contraction induced by calyculin-A was not affected by SP600125. On the other hand, JNKs were phosphorylated by CCh (2.2 ± 0,4 [mean±SEM] fold increase). The JNK phosphorylation induced by CCh was significantly inhibited by SP600125 (n = 4). These findings suggest that the Tyr kinase/JNK pathway may play a role in bronchial smooth muscle contraction. Strategies to inhibit JNK activation may represent a novel therapeutic approach for diseases involving airway obstruction, such as asthma and chronic obstructive pulmonary disease.

Effects of hydrogen sulphide on motility patterns in the rat colon

Hydrogen sulphide (H2 S) is an endogenous gaseous signalling molecule with putative functions in gastrointestinal motility regulation. Characterization of H2 S effects on colonic motility is crucial to establish its potential use as therapeutic agent in the treatment of colonic disorders. H2 S effects on colonic motility were characterized using video recordings and construction of spatio-temporal maps. Microelectrode and muscle bath studies were performed to investigate the mechanisms underlying H2 S effects. NaHS was used as the source of H2 S. Rhythmic propulsive motor complexes (RPMCs) and ripples were observed in colonic spatio-temporal maps. Serosal addition of NaHS concentration-dependently inhibited RPMCs. In contrast, NaHS increased amplitude of the ripples without changing their frequency. Therefore, ripples became the predominant motor pattern. Neuronal blockade with lidocaine inhibited RPMCs, which were restored after administration of carbachol. Subsequent addition of NaHS inhibited RPMCs. Luminal addition of NaHS did not modify motility patterns. NaHS inhibited cholinergic excitatory junction potentials, carbachol-induced contractions and hyperpolarized smooth muscle cells, but did not modify slow wave activity. H2 S modulated colonic motility inhibiting propulsive contractile activity and enhancing the amplitude of ripples, promoting mixing. Muscle hyperpolarization and inhibition of neurally mediated cholinergic responses contributed to the inhibitory effect on propulsive activity. H2 S effects were not related to changes in the frequency of slow wave activity originating in the network of interstitial cells of Cajal located near the submuscular plexus. Luminal H2 S did not modify colonic motility probably because of epithelial detoxification.

Anti-diarrheal constituents of Alpinia oxyphylla

Isolation, screening and in vivo assays have been used for evaluating anti-diarrhea bioactive of Alpinia oxyphylla. Preliminary experimental results showed that 95% ethanol extract and 90% ethanol elution significantly extended the onset time of diarrhea and reduced the wet feces proportion, however 50% ethanol election had no effect on diarrhea. Chemical analysis results displayed that Nootkatone, Tectochrysin and yakuchinone A may be bioactive ingredients for curing diarrhea. Duodenum in vitro experiment showed that Tectochrysin 50, 100μM reduces carbachol-induced contraction, while yakuchinone A and Nootkatone had no effect. Bioinformatic computational method as molecular docking has been complementary to experimentally work to explore the potential mechanism. The study of pathogenesis of diarrhea in humans and animal models suggested that Na+/H+ exchanger3 (NHE3) and aquaporin4 (AQP4) are causative agents of diarrhea. The analysis was done on the basis of scoring and binding ability and the docking analysis showed that Tectochrysin has maximum potential against NHE3 (PDB ID: 2OCS) and AQP4 (PDB ID: 3GD8). Tectochrysin indicated minimum energy score and the highest number of interactions with active site residues. These results suggested that A. oxyphylla might exhibit its anti-diarrhea effect partially by affecting the proteins of NHE3 and AQP4 with its active ingredient Tectochrysin.

PTHrP Is Endogenous Relaxant for Spontaneous Smooth Muscle Contraction in Urinary Bladder of Female Rat

Acute bladder distension causes various morphologic and functional changes, in part through altered gene expression. We aimed to investigate the physiologic role of PTHrP, which is up-regulated in an acute bladder distension model in female rats. In the control Empty group, bladders were kept empty for 6 hours, and in the Distension group, bladders were kept distended for 3 hours after an artificial storing-voiding cycle for 3 hours. In the Distention group bladder, up-regulation of transcripts was noted for 3 genes reported to be up-regulated by stretch in the cultured bladder smooth muscle cells in vitro. Further transcriptome analysis by microarray identified PTHrP as the 22nd highest gene up-regulated in Distension group bladder, among more than 27,000 genes. Localization of PTHrP and its functional receptor, PTH/PTHrP receptor 1 (PTH1R), were analyzed in the untreated rat bladders and cultured bladder cells using real-time RT-PCR and immunoblotting, which revealed that PTH1R and PTHrP were more predominantly expressed in smooth muscle than in urothelium. Exogenous PTHrP peptide (1-34) increased intracellular cAMP level in cultured bladder smooth muscle cells. In organ bath study using bladder strips, the PTHrP peptide caused a marked reduction in the amplitude of spontaneous contraction but caused only modest suppression for carbachol-induced contraction. In in vivo functional study by cystometrogram, the PTHrP peptide decreased voiding pressure and increased bladder compliance. Thus, PTHrP is a potent endogenous relaxant of bladder contraction, and autocrine or paracrine mechanism of the PTHrP-PTH1R axis is a physiologically relevant pathway functioning in the bladder.

Involvement of Connexins 43 and 45 in Functional Mechanism of Human Detrusor Overactivity in Neurogenic Bladder

To assess the involvement of connexins (Cxs) 43 and 45 in the selective inhibition of detrusor contractions in patients with neurogenic detrusor overactivity (NDO). Detrusor strips with and without mucosa were obtained from patients undergoing cystectomy for either neurogenic bladder with NDO refractory to pharmacologic treatment or bladder cancer with no overactive bladder symptoms (ie, control group). The strips were isometrically mounted in organ baths. The effects of the selective inhibitors Cxs 43 and 45 on carbachol-induced contractions of bladder strips were assessed. Overall, 47 patients with a median age of 61 years (range 19-83) were included. The female-to-male ratio was 0.42. Selective inhibitors of Cxs 43 and 45 significantly inhibited carbachol-induced contractions of bladder strips from patient with NDO (P <.01) but had no effect in the control group. When tested without mucosa, the effect of the Cx 43 inhibitor in the NDO patient strips was abolished, but it remained unchanged with the Cx 45 inhibitor (P <.05). The pharmacologic modulation of Cx-mediated intercellular communication, targeting Cxs 43 and 45, is directly involved in the functional mechanism of NDO and might represent a future target for the treatment of NDO.

A role for M2 and M3 muscarinic receptors in the contraction of rat and human small airways

Large airway bronchoconstriction acts mainly through cholinergic pathways via muscarinic M3 receptors with some contribution from M2 receptors. By contrast, the mechanisms of small airway contraction are largely unknown. This study used precision cut lung slices to examine the role of muscarinic M2 and M3 receptors in the contractile response of rat and human small airways.In rat small airways, the M3 preferential antagonist, 4-DAMP, inhibited carbachol-mediated contraction (1×10−6M) more than that of the M2 selective antagonist, AF-DX116 (pIC50 values: 8.85±0.18 and 6.31±0.19, n=6–8 respectively). Tiotropium, inhibited the contractile response to carbachol with (pIC50: 9.86±0.07, n=6), but could not distinguish between M2 and M3 mediated effects. Similar experiments using human small airways with tiotropium and AF-DX116, gave a pIC50 of 10.35±0.05 and a pKB of 6.37±0.13, n=5 respectively. Therefore, M3 receptors play a key role in muscarinic contraction of small airways in both rats and humans but the effect of M2 receptors cannot be excluded.To investigate the role of M2 receptors, carbachol-induced contraction of small airways was performed in the presence and absence of a β2-agonist in order to elevate intracellular cAMP levels prior to contraction. Isoproterenol-induced relaxation was significantly increased by AF-DX116 (P<0.001) in rat small airways and by AF-DX116 (P<0.01), gallamine (P<0.05) and pertussis toxin (P<0.05) in human small airways.Taken together, these data suggest that cholinergic antagonism of muscarinic receptors in human and rat small airways inhibits airway contraction via direct inhibition of contraction through M3 receptors, and by M2 receptor mediated inhibition of relaxation.

Signaling cross-talk between cannabinoid and muscarinic systems actives Rho-kinase and increases the contractile responses of the bovine ciliary muscle

The aim of the present study was to evaluate the role of a possible interaction between cannabinoid and muscarinic systems, both widely expressed in the ocular structure and involved in the control of bovine ciliary muscle contractility and intraocular pressure modulation. The ciliary muscle strips isolated by bovine eyes were exposed cumulatively to anandamide in the presence and in the absence of carbachol (5nM), in a miograph system for isometric recording. The experiments were also conducted in the presence of AM251 (100nM), 4-DAMP (100nM), Pertussis toxin (500ng/ml), U73122 (0.1 and 1μM), chelerythrine (1 and 10μM) and Y27632 (1 and 10μM). Contractile responses were expressed as the percentage of 10μM carbachol-induced contraction. The anandamide-induced contraction on bovine ciliary muscle strips was enhanced by the previous stimulation of Gq-protein-coupled muscarinic M3 receptors with carbachol. The contractile response to anandamide plus carbachol was affected by different inhibitors such as Pertussis toxin, phospholipase C, protein kinase C and Rho-kinase. The key results of the present study show that sequential activation of muscarinic M3 receptors and cannabinoid CB1 receptors produce synergistic contractile effects of the bovine ciliary muscle by involving the activation of Rho-kinase and protein kinase C.

TRPM8 agonists modulate contraction of the pig urinary bladder

The transient receptor potential melastin-8 (TRPM8) channel is activated by the "cooling" compounds menthol and icilin. Pathophysiologically, it is implicated in the overactive bladder and bladder cooling reflex, but the activity of TRPM8 in normal bladder physiology is poorly understood. We investigated the distribution of TRPM8 channels and the effect of TRPM8 agonists on the contractile function of pig bladder (n = 35) strips and whole bladders. The distribution of TRPM8 was examined by immunohistochemistry. The effect of vesical or intravascular menthol (0.1-0.3 mmol/L) or icilin (50 μmol/L) on carbachol-induced isolated whole bladder contractions was monitored by recording vesical pressure. Strips of denuded detrusor or mucosa were mounted in organ baths to study the effect of TRPM8 agonists on the contractile responses to 10 μmol/L carbachol. TRPM8-like immunoreactivity was detected on pig urothelium. Intravascular menthol (0.3 mmol/L) and icilin (50 μmol/L) significantly decreased the magnitude of carbachol-induced whole bladder contraction, whereas vesical administration significantly increased the response. In detrusor and mucosal strips, both menthol (0.3 mmol/L) and icilin (50 μmol/L) inhibited carbachol-induced contractions. We conclude that the TRPM8 channel is expressed on the urothelium of pig bladder. In the whole organ, exposure of the urothelium to menthol or icilin increases the contractile response to carbachol. Where detrusor muscle is exposed directly to these compounds, the contractile response to carbachol is reduced.

Activation of G protein-coupled bile acid receptor, TGR5, induces smooth muscle relaxation via both Epac- and PKA-mediated inhibition of RhoA/Rho kinase pathway

The present study characterized the TGR5 expression and the signaling pathways coupled to this receptor that mediates the relaxation of gastric smooth muscle. TGR5 was detected in gastric muscle cells by RT-PCR and Western blotting. Treatment of cells with the TGR5-selective ligand oleanolic acid (OA) activated Gαs, but not Gαq, Gαi1, Gαi2, or Gαi3, and increased cAMP levels. OA did not elicit contraction, but caused relaxation of carbachol-induced contraction of gastric muscle cells from wild-type mice, but not tgr5(-/-) mice. OA, but not a selective exchange protein activated by cAMP (Epac) ligand (8-pCPT-2'-O-Me-cAMP), caused phosphorylation of RhoA and the phosphorylation was blocked by the PKA inhibitor, myristoylated PKI, and by the expression of phosphorylation-deficient mutant RhoA (S188A). Both OA and Epac ligand stimulated Ras-related protein 1 (Rap1) and inhibited carbachol (CCh)-induced Rho kinase activity. Expression of RhoA (S188A) or PKI partly reversed the inhibition of Rho kinase activity by OA but had no effect on inhibition by Epac ligand. However, suppression of Rap1 with siRNA blocked the inhibition of Rho kinase by Epac ligand, and partly reversed the inhibition by OA; the residual inhibition was blocked by PKI. Muscle relaxation in response to OA, but not Epac ligand, was partly reversed by PKI. We conclude that activation of TGR5 causes relaxation of gastric smooth muscle and the relaxation is mediated through inhibition of RhoA/Rho kinase pathway via both cAMP/Epac-dependent stimulation of Rap1 and cAMP/PKA-dependent phosphorylation of RhoA at Ser(188). TGR5 receptor activation on smooth muscle reveals a novel mechanism for the regulation of gut motility by bile acids.

A Novel Muscarinic Antagonist R2HBJJ Inhibits Non-Small Cell Lung Cancer Cell Growth and Arrests the Cell Cycle in G0/G1

Lung cancers express the cholinergic autocrine loop, which facilitates the progression of cancer cells. The antagonists of mAChRs have been demonstrated to depress the growth of small cell lung cancers (SCLCs). In this study we intended to investigate the growth inhibitory effect of R2HBJJ, a novel muscarinic antagonist, on non-small cell lung cancer (NSCLC) cells and the possible mechanisms. The competitive binding assay revealed that R2HBJJ had a high affinity to M3 and M1 AChRs. R2HBJJ presented a strong anticholinergic activity on carbachol-induced contraction of guinea-pig trachea. R2HBJJ markedly suppressed the growth of NSCLC cells, such as H1299, H460 and H157. In H1299 cells, both R2HBJJ and its leading compound R2-PHC displayed significant anti-proliferative activity as M3 receptor antagonist darifenacin. Exogenous replenish of ACh could attenuate R2HBJJ-induced growth inhibition. Silencing M3 receptor or ChAT by specific-siRNAs resulted in a growth inhibition of 55.5% and 37.9% on H1299 cells 96 h post transfection, respectively. Further studies revealed that treatment with R2HBJJ arrested the cell cycle in G0/G1 by down-regulation of cyclin D1-CDK4/6-Rb. Therefore, the current study reveals that NSCLC cells express an autocrine and paracrine cholinergic system which stimulates the growth of NSCLC cells. R2HBJJ, as a novel mAChRs antagonist, can block the local cholinergic loop by antagonizing predominantly M3 receptors and inhibit NSCLC cell growth, which suggest that M3 receptor antagonist might be a potential chemotherapeutic regimen for NSCLC.

Muscarinic receptor M3 mediates human gallbladder contraction through voltage-gated Ca2+ channels and Rho kinase

Objective. Muscarinic receptors mediate contraction of the human gallbladder through unclear receptor subtypes. The aim of the present study was to characterize muscarinic acetylcholine receptors mediating contraction of the human gallbladder. Materials and methods. Contraction of human gallbladder muscle strips caused by agonists carbachol and muscarine was measured and the inhibition of carbachol-induced contraction by muscarinic receptor antagonists was evaluated. Reverse transcription polymerase chain reaction was performed to determine the existence of muscarinic receptor subtypes. Results. Carbachol and muscarine caused concentration-dependent contraction of gallbladder strips. Four receptor antagonists, including atropine, 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP), methoctramine, and pirenzepine, inhibited the carbachol-induced contraction. The relative inhibitory potency of these receptor antagonists was atropine > 4-DAMP > methoctramine > pirenzepine. The antagonist affinity estimates (pA2 values) correlated with the known affinities at M3, M4, and M5 muscarinic receptors. In addition, the M4-selective antagonist muscarinic toxin 3 did not inhibit and the M5-selective positive allosteric modulator VU0238429 did not potentiate carbachol-induced gallbladder contraction. This suggests that M3 muscarinic receptors mediate the muscarinic response predominantly. The contractile response of carbachol was attenuated by the voltage-gated Ca2+ channel inhibitor nifedipine and Rho-kinase inhibitor H-1152, but not affected by protein kinase C inhibitor chelerythrine. This implies the involvement of voltage-gated Ca2+ channel and Rho kinase but not protein kinase C. Conclusions. These results suggest a major role of M3 muscarinic receptors mediating the human gallbladder contraction through voltage-gated Ca2+ channels and Rho kinase. M3-selective muscarinic receptor antagonists could be of therapeutic importance in the treatment of biliary motility disorders.

(−)-Carvone: Antispasmodic effect and mode of action

(−)-Carvone is a monoterpene ketone found in spearmint (Mentha spicata var. crispa) essential oil that is widely used as an odor and flavor additive. An intestinal antispasmodic effect was recently reported for (−)-carvone, and it has been shown to be more potent than its (+)-antipode. The mechanism of (−)-carvone action in the intestines has not been investigated. To gain a better understanding of the (−)-carvone antispasmodic effect, we investigated its pharmacological effects in the guinea pig ileum. Terminal portions of the ileum were mounted for isotonic contraction recordings. The effect of (−)-carvone was compared with that of the classical calcium channel blocker (CCB) verapamil. In isolated ileal smooth muscle, (−)-carvone did not produce direct contractile or relaxation responses and did not modify electrically elicited contractions or low K+-evoked contractions. The submaximal contractions induced by histamine (p<0.001), BaCl2 (p<0.05), and carbachol (p<0.01) were significantly reduced by (−)-carvone. The contractile response elicited by high concentrations of carbachol was reduced but not abolished by (−)-carvone. No additive action was detected with co-incubation of (−)-carvone and verapamil on carbachol-induced contraction. (−)-Carvone reduced the contraction induced by high K+ and was almost 100 times more potent than verapamil. Thus, (−)-carvone showed a typical and potent CCB-like action. Many effects described for both (−)-carvone and spearmint oil can be explained as a CCB-like mode of action.

The effect of hypercholesterolemia on carbachol-induced contractions of the detrusor smooth muscle in rats: increased role of L-type Ca2+ channels

To investigate a possible relation between hypercholesterolemia and detrusor smooth muscle function, we studied the contractile response to potassium challenge, carbachol (CCh), and the components of CCh-induced contractile mechanism in high-cholesterol diet-fed rats. Adult male Sprague-Dawley rats were fed with standard (control group, N = 17) or 4% cholesterol diet (hypercholesterolemia group (HC), N = 16) for 4weeks. Spontaneous contractions of detrusor muscle strips and their responses to potassium chloride (KCl) or cumulative dose-contraction curves to CCh were recorded. The effects of muscarinic receptor antagonists (methoctramin and/or 4-diphenylacetoxy-N-methylpiperidine), L-type Ca(+2) channel blocker (nifedipine), and/or rho-kinase inhibitor Y-27632 were investigated. Blood cholesterol level was increased in the HC group with no sign of atherosclerosis. The KCl-induced detrusor smooth muscle contractions were higher in HC, whereas spontaneous and CCh-induced responses were similar in both groups. Preincubation with receptor antagonist for M(3) but not for M(2) attenuated contraction significantly, shifting the dose-response curve to the right. This response was similar in both groups. Among two effector mechanisms of M(3)-mediated detrusor smooth muscle contraction, rho-kinase pathway was not affected by hypercholesterolemia, whereas blockade of L-type Ca(+2) channels potently reduced contractions. The results of this study point out a relation between hypercholesterolemia and contractile mechanism of detrusor smooth muscle likely to change urinary bladder function, via altering L-type Ca(+2) channels. Taken together with escalating incidence of hypercholesterolemia and lower urinary tract symptoms, it is a field which deserves to be investigated further.

Long-acting formulation of a new muscarinic receptor antagonist for the treatment of overactive bladder

A new muscarinic receptor antagonist, 5-hydroxymethyl tolterodine (5-HMT), was successfully encapsulated into PLGA microspheres. With an increase of PLGA concentration from 15% to 40%, encapsulation efficiency of 5-HMT increased from 55.39% to 76.32%, and the particle size of microsphere increased from 34.33 to 70.48 µm. Increasing the homogenisation speed from 850 to 2300 rpm, the particle size was reduced about 65%.The in vitro and in vivo studies in beagle dogs show that the release profile of 5-HMT-loaded microspheres (5-HMT MS) prepared with 503H is characterised by a low initial burst followed by slow release that lasted for 2 weeks. A Cmax of 1.617 ± 0.392 ng/mL was found on the sixth day. When evaluated for inhibition of the carbachol-induced contraction of rat urinary bladder, 5-HMT MS showed a much longer and more potent effect than tolterodine tablets. The mean urination time of the rats in the 5-HMT MS group was significantly decreased (p < 0.05 or p < 0.01) to less than 2 weeks.

Expression of alternatively spliced variants of Na–Ca-exchanger-1 in experimental colitis: role in reduced colonic contractility

Inflammation-induced colonic motility dysfunction is associated with a disturbance in Ca(2+) ion transporting mechanisms. The main objective of this study was to identify the types of Na-Ca-exchanger-1 (NCX-1) variants expressed in the rat colon, and how this was affected by colitis. In addition, the effect of colitis on the possible involvement of NCX-1 in the reduced carbachol-induced contraction of the rat colon was examined. Colitis was induced in male Sprague-Dawley rats by intra-rectal instillation of trinitrobenzenesulphonic acid (TNBS). Animals were killed on day 5. Colitis was characterized by estimating myeloperoxidase (MPO) activity, body weight, and histological scores. NCX-1 mRNA and protein variants were confirmed by RT-PCR coupled nucleotide sequencing and by Western blot analysis, respectively. Contractility of the colon segments was studied using standard procedure. There was a significant reduction in body weight of TNBS-treated rats. A significant increase in MPO activity and infiltration of inflammatory cells were observed in the inflamed rat colon. RT-PCR coupled nucleotide sequencing identified NCX-1.3 mRNA variant containing exons B and D. Western blot analysis confirmed 70 and 120 kDa molecular mass NCX-1 protein variants in rat colon. There was no significant difference (p > 0.05) in the level of NCX-1 protein variants in inflamed colon as compared to non-colitis controls. Functional experiments demonstrated that NCX in reverse mode played a role in carbachol-induced contraction of colon, and this was not affected by colitis. These findings demonstrated expression of a NCX-1.3 mRNA splice variant, and 70 and 118 kDa protein variants. Inhibition of the reverse mode of NCX-1 was not different in reduced carbachol-induced contraction between the groups. These findings are interpreted to suggest that NCX-1, though expressed did not play a role in reduced contractility in experimental colitis.

Bryophyllum pinnatum inhibits detrusor contractility in porcine bladder strips—A pharmacological study towards a new treatment option of overactive bladder

AimsA broad spectrum of synthetic agents is available for the treatment of overactive bladder. Anti-cholinergic drugs show a poor compliance due to side effects. There is an increasing use of plant extracts in medicine. We have therefore investigated the inhibitory effects of leaf press juice from Bryophyllum pinnatum (Lam.) Oken (Kalanchoe pinnata L.) on bladder strips and compared the effects to that of oxybutynin. MethodsStrips of porcine detrusor were prepared in Krebs solution and contractility was measured in a myograph system chamber aired with O2/CO2 at 37°C. To induce contractions, electrical field stimulation (32Hz, 40V) was used for the inhibitory effect measurements, and carbachol (50μM) for the relaxant effect measurements. Recordings were obtained in the absence and presence of increasing concentrations of Bryophyllum pinnatum leaf press juice (BPJ, 0.1–10%), and oxybutynin (10−7–10−3M) as a reference substance. ResultsIn inhibition experiments, BPJ as well as oxybutynin inhibited electrically induced contractions of porcine detrusor. BPJ at concentrations of 5% inhibited the contraction compared to a time matched control significantly by 74.6±10.2% (p<0.001). BPJ as well as oxybutynin relaxed carbachol pre-contracted porcine detrusor strips. The maximum relaxant effect of BPJ compared to a time matched control was 18.7±3.7 (p<0.05) at a concentration of 10% BPJ. ConclusionsOur investigations show that BPJ inhibits contractions induced by electrical field stimulation and relaxes carbachol-induced contractions. However, the effect was lower than that of the reference substance oxybutynin. It is important to continue in vitro experiments as well as clinical studies with BPJ that might offer a new treatment option for patients with OAB.

Characteristics of AZD9708, a novel, selective β2-adrenoceptor agonist with rapid onset and long duration of action

Here we describe the pre-clinical pharmacological profile of AZD9708, a novel long-acting β2-adrenoceptor agonist that has potential as a once-daily therapy for asthma and chronic obstructive pulmonary disease (COPD).AZD9708 is a potent and selective agonist at the human β2-adrenoceptor, with selectivity over human β1- and β3-adrenoceptors of >500 and >24 fold, respectively. AZD9708 relaxes carbachol-induced contraction of human bronchial rings with a time to 90% of maximal relaxation of 13–20 min, similar to that seen with formoterol and quicker than salmeterol. In anesthetized guinea pigs, AZD9708 provides significant protection against histamine-induced airway constriction at 24 h after intratracheal and nebulized doses. This is longer than with intratracheal salmeterol, which is bronchoprotective for approximately 8 h, and formoterol, which is bronchoprotective for 8 and 12 h following nebulized and intratracheal dosing, respectively.AZD9708 also shows the potential for a greater therapeutic margin than widely used β2-adrenoceptor agonists such as formoterol. At a defined efficacy dose that provides 80% bronchoprotection (ED80), formoterol leads to a decrease in blood potassium levels in guinea pigs, whilst AZD9708 is not associated with significant reductions in potassium levels at doses up to 7 times the ED80. [14C]AZD9708 is associated with extensive protein binding in both human (mean 1.0% free) and rat (mean 2.6% free) plasma.This pharmacological profile indicates the potential of AZD9708 to become an important addition to the range of bronchodilators available for the treatment of patients with obstructive airways disease.

Effect of silymarin on bladder overactivity in cyclophosphamide-induced cystitis rat model

The purpose of this study was to investigate the effects of silymarin, a phytotherapeutic agent, on bladder overactivity in a cyclophosphamide (CYP)-induced cystitis rat model. Female Wistar Albino rats received a single intraperitoneal injection of CYP (150mg/kg) or saline and after 72h, bladder function was evaluated by in vitro preparations of whole bladders and cystometry with continuous saline infusion under urethane anesthesia. Silymarin or a vehicle was orally given for 7 days in rats. CYP was injected on the 5th day of silymarin or vehicle treatment and then the animals were killed on the 8th day. CYP-treatment dramatically potentiated the basal spontaneous contractions of isolated whole bladders compared to control rats. In anesthetized rats, during continuous infusion cystometry, intercontraction interval (ICI) was significantly shorter, but bladder voiding pressure was not significantly changed in CYP-injected rats compared to control rats. In the CYP-injected group, silymarin treatment significantly decreased the amplitude, frequency (contractions/min) and area under the curve of spontaneous contractions, but failed to change carbachol-induced contraction in isolated whole bladder. Also, silymarin treatment significantly increased the ICI in comparison to the vehicle treatment. In the saline-injected group, no significant changes in the bladder function were observed between the silymarin and vehicle-treated groups. Histopathological examination showed that CYP-induced bladder inflammation tended to be lower in the silymarin+CYP-treated group. In conclusion, the oral administration of silymarin suppressed CYP-induced bladder overactivity. Silymarin may be considered as an attractive treatment for CYP-induced bladder overactivity.

Inhibition of phosphodiesterases relaxes detrusor smooth muscle via activation of the large-conductance voltage- and Ca2+-activated K+ channel

Detrusor smooth muscle (DSM) exhibits increased spontaneous phasic contractions under pathophysiological conditions such as detrusor overactivity (DO). Our previous studies showed that activation of cAMP signaling pathways reduces DSM contractility by increasing the large-conductance voltage- and Ca(2+)-activated K(+) (BK) channel activity. Here, we tested the hypothesis whether inhibition of phosphodiesterases (PDEs) can reduce guinea pig DSM excitability and contractility by increasing BK channel activity. Utilizing isometric tension recordings of DSM isolated strips and the perforated patch-clamp technique on freshly isolated DSM cells, we examined the mechanism of DSM relaxation induced by PDE inhibition. Inhibition of PDEs by 3-isobutyl-1-methylxanthine (IBMX), a nonselective PDE inhibitor, significantly reduced DSM spontaneous and carbachol-induced contraction amplitude, frequency, duration, muscle force integral, and tone in a concentration-dependent manner. IBMX significantly reduced electrical field stimulation-induced contractions of DSM strips. Blocking BK channels with paxilline diminished the inhibitory effects of IBMX on DSM contractility, indicating a role for BK channels in DSM relaxation mediated by PDE inhibition. IBMX increased the transient BK currents (TBKCs) frequency by ∼3-fold without affecting the TBKCs amplitude. IBMX increased the frequency of the spontaneous transient hyperpolarizations by ∼2-fold and hyperpolarized the DSM cell resting membrane potential by ∼6 mV. Blocking the BK channels with paxilline abolished the IBMX hyperpolarizing effects. Under conditions of blocked Ca(2+) sources for BK channel activation, IBMX did not affect the depolarization-induced steady-state whole cell BK currents. Our data reveal that PDE inhibition with IBMX relaxes guinea pig DSM via TBKCs activation and subsequent DSM cell membrane hyperpolarization.

Two for one: Cyclic AMP mediates the anti-inflammatory and anti-spasmodic properties of the non-anesthetic lidocaine analog JMF2-1

Inhalation of JMF2-1, an analog of lidocaine with reduced anesthetic activity, prevents airway contraction and lung inflammation in experimental asthma models. We sought to test if the JMF2-1 effects are a consequence of increased intracellular cAMP levels in asthma cell targets, such as smooth muscle cells and T cells. Functional effect of JMF2-1 on carbachol-induced contraction of intact or epithelial-denuded rat trachea was assessed in conventional organ baths. cAMP was quantified by radioimmunoassay in cultured guinea pig tracheal smooth muscle cells, as well as lymph node cells from BALB/c mice, exposed to JMF2-1. We found that JMF2-1 (0.1–1mM) concentration-dependently inhibited epithelium-intact tracheal ring contraction induced by carbachol challenge. The antispasmodic effect remained unaltered following epithelium removal or pretreatment with NG-nitro-L-arginine methyl ester (100μM), but it was clearly sensitive to 9-(tetrahydro-2-furyl) adenine (SQ22,536, 100μM), an adenylate cyclase inhibitor. JMF2-1 (300 and 600μM) also dose-dependently increased cAMP intracellular levels of both cultured airway smooth muscle cells and T lymphocytes. This effect was consistently abrogated by SQ22,536 and reproduced by forskolin in both systems. JMF2-1 induced apoptosis of anti-CD3 activated T cells in a mechanism sensitive to zIETD, indicating that JMF2-1 mediates caspase-8-dependent apoptosis. Furthermore, forskolin also inhibited anti-CD3 induced T cell proliferation and survival. Our results suggest that JMF2-1 inhibits respiratory smooth muscle contraction as well as T cell proliferation and survival through enhancement of intracellular cAMP levels. These findings may help to explain the anti-inflammatory and antispasmodic effects of JMF2-1 observed in previous studies.