Carbachol-induced Contraction Research Articles

Diffusion of nitric oxide through the gastric wall upon reduction of nitrite by red wine: Physiological impact

In this work we showed that nitric oxide produced via red wine- and ascorbate-dependent reduction of nitrite diffuses through the rat stomach, inducing smooth muscle relaxation. The studies encompassed ex vivo and in vivo models of diffusion. Regarding the former, luminal NO generated from a mixture of physiologic nitrite and ascorbate or wine diffuses across the stomach wall, being 8–20% of that produced in the mucosal side detected at high μM range (>100 μM) in the serosal side. In order to evaluate whether cellular dysfunction was associated with NO diffusion at the μM range, the gastric tissue exposed to NO was evaluated in terms of carbachol-induced muscle contraction in fundal strips and mitochondrial respiration and showed to remain functional and metabolically active. Moreover, pre-contracted gastric strips were shown to relax 86.5 ± 5.5% (control) and 75.0 ± 4.0% (nitrite/ascorbate-exposed tissue) when challenged with acidified nitrite. The studies in the living animal support the diffusion of luminal NO to the gastric vasculature as, following addition of nitrite/ascorbate to rat stomach in vivo, NO was not detected in the serosal environment but concentrations as high as 31 μM of NO were detected outside the stomach after cardiac arrest. Collectively, the results establish a link between the consumption of nitrite and dietary reductants (e.g., wine polyphenols) and stomach muscle relaxation via the local chemical generation of NO.

Inhibitory effect of quercetin on rat trachea contractility in vitro

Abstract Objectives The effect of quercetin, a naturally occurring flavonoid traditionally used to treat airway diseases such as bronchial asthma, on the contractile response elicited by electrical field stimulation or carbachol in rat isolated trachea was investigated. Methods Isolated tracheal tissue was subjected to contractions by an electrical field stimulation of 5 Hz for 30 s, 400 mA, and the responses in the presence of cumulative concentrations of quercetin (10−6−3 × 10−4 M) were observed. The effect of quercetin was also evaluated after administration of phentolamine plus propranolol (to block α- and β-adrenergic receptors), NG-nitro-L-arginine methyl ester (to block nitric oxide synthesis), capsaicin (to desensitise sensory C fibres), α-chymotrypsin (a proteolytic enzyme that rapidly degrades vasoactive intestinal peptide), SR140333 and SR48968 (tackykinin NK1 and NK2 receptor antagonists, respectively). Key findings Quercetin produced a concentration-dependent inhibition of contractions induced by both carbachol and electrical field stimulation. However, quercetin was more active in inhibiting the contractions produced by electrical field stimulation than those induced by carbachol, suggesting a presynaptic site of action (in addition to a postsynaptic effect, as revealed by the inhibitory action of quercetin on carbachol-induced contractions). The inhibitory effect of quercetin on contractions induced by electrical field stimulation was unaffected by phentolamine plus propranolol, SR 140333 and SR 48968, capsaicin treatment or by the proteolytic enzyme α-chymotrypsin. In contrast, the nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester significantly reduced the inhibitory effect of quercetin on contractions induced by electrical field stimulation. Conclusions Quercetin inhibits rat tracheal contractility through a presynaptic (involving nitric oxide) and a postsynaptic site of action.

Effect of Four Medications Associated with Gastrointestinal Motility on Oddi Sphincter in the Rabbit

Aim: Modulatory drugs of gastrointestinal (GI) motility are a possibility for use to relieve the main clinical presentation of sphincter of Oddi (SO) dysfunctions which are not easily distinguished from those occurring in high prevalence functional GI disorders. The aim of this study was to investigate the effects of GI motility modulators including pinaverium, domperidone, trimebutine, and tegaserod on the contractile activity of SO stimulated by carbachol in the rabbit. Methods: The contraction responses precontracted by carbachol (0.1 μM) of in vitro rabbit SO rings were evaluated before and after the addition of a series concentration (10−13 to 10−3 M) of pinaverium, domperidone, trimebutine, and tegaserod. Results: Pinaverium induced a concentrationdependent relaxation of isolated SO rings (10−13 vs. 10−7 vs. 10−13 M = 16.6 ± 4.8 vs. 47.1 ± 5.5 vs. 81.2 8 6.2%, p<0.001 by ANOVA) precontracted with carbachol (0.1μM). Tegaserod did not significantly effect (10−13 vs. 10−7 vs. 10−3 M = 2.3 ± 2.2 vs. 6.7± 2.1 vs. 10.1 ± 2.3%, p>0.05 by ANOVA) SO motility, but domperidone seemed to stimulate SO contractions (1010−12 vs. 1010−7 vs. 1010−3 M = −2.2 ± 1.5 vs. −13.9 ± 2.0 vs. −21.0 ± 2.7%, p<0.05 by ANOVA). At low doses (10−13 to 10−7 M), trimebutine stimulated SO contraction (−8.7 ± 1.4 vs. −9.3 ± 2.0%); however, high doses (10−6 to 10−3 M) of trimebutine inhibited SO motility (−-5.9 ± 1.7 vs. 14.5 ±2.0%, p<0.05 by ANOVA). Conclusion: Pinaverium totally inhibits contractions induced by carbachol and tegaserod has no effect on carbachol-induced contractions. Domperidone stimulates contractions induced by carbachol. Trimebutine could either stimulate or inhibit SO contractions depending on its dosage.

MP-02.06: Effects of Antimuscarinic Agents on Carbachol-Induced Contraction of Normal Human Detrusor and Overactive Detrusor Associated with Benign Prostatic Obstruction

The present study was performed to investigate whether the antagonist effect of various antimuscarinic agents on carbachol-induced contraction differed between normal human detrusor muscle and detrusor overactivity (DO) in patients with benign prostatic obstruction (BPO).

Carbachol-induced rabbit bladder smooth muscle contraction: roles of protein kinase C and Rho kinase

Smooth muscle contraction is regulated by phosphorylation of the myosin light chain (MLC) catalyzed by MLC kinase and dephosphorylation catalyzed by MLC phosphatase. Agonist stimulation of smooth muscle results in the inhibition of MLC phosphatase activity and a net increase in MLC phosphorylation and therefore force. The two pathways believed to be primarily important for inhibition of MLC phosphatase activity are protein kinase C (PKC)-catalyzed CPI-17 phosphorylation and Rho kinase (ROCK)-catalyzed myosin phosphatase-targeting subunit (MYPT1) phosphorylation. The goal of this study was to determine the roles of PKC and ROCK and their downstream effectors in regulating MLC phosphorylation levels and force during the phasic and sustained phases of carbachol-stimulated contraction in intact bladder smooth muscle. These studies were performed in the presence and absence of the PKC inhibitor bisindolylmaleimide-1 (Bis) or the ROCK inhibitor H-1152. Phosphorylation levels of Thr(38)-CPI-17 and Thr(696)/Thr(850)-MYPT1 were measured at different times during carbachol stimulation using site-specific antibodies. Thr(38)-CPI-17 phosphorylation increased concurrently with carbachol-stimulated force generation. This increase was reduced by inhibition of PKC during the entire contraction but was only reduced by ROCK inhibition during the sustained phase of contraction. MYPT1 showed high basal phosphorylation levels at both sites; however, only Thr(850) phosphorylation increased with carbachol stimulation; the increase was abolished by the inhibition of either ROCK or PKC. Our results suggest that during agonist stimulation, PKC regulates MLC phosphatase activity through phosphorylation of CPI-17. In contrast, ROCK phosphorylates both Thr(850)-MYPT1 and CPI-17, possibly through cross talk with a PKC pathway, but is only significant during the sustained phase of contraction. Last, our results demonstrate that there is a constitutively activate pool of ROCK that phosphorylates MYPT1 in the basal state, which may account for the high resting levels of MLC phosphorylation measured in rabbit bladder smooth muscle.

HSP20 phosphorylation and airway smooth muscle relaxation

HSP20 (HSPB6) is a small heat shock protein expressed in smooth muscles that is hypothesized to inhibit contraction when phosphorylated by cAMP-dependent protein kinase. To investigate this hypothesis in airway smooth muscle (ASM) we showed that HSP20 was constitutively expressed as well as being inducible in cultured hASM cells by treatment with 1 μM isoproterenol or 10 μM salmeterol. In contrast, a mixture of proinflammatory mediators (interleukin-1β, tumor necrosis factor α, and interferon γ) inhibited expression of HSP20 by about 50% in 48 hours. To determine whether phosphorylation of HSP20 is sufficient to induce relaxation, canine tracheal smooth muscle was treated with a cell permeant phosphopeptide that mimics the phosphorylation of HSP20. The HSP20 phosphopeptide antagonized carbachol-induced contraction by 60% with no change in myosin light chain phosphorylation. Recombinant full length HSP20 inhibited skeletal actin binding to smooth muscle myosin subfragment 1 (S1), and recombinant cell permeant TAT-HSP20 S16D mutant reduced F-actin filaments in cultured hASM cells. Carbachol stimulation of canine tracheal smooth muscle tissue caused redistribution of HSP20 from large macromolecular complexes (200-500 kDa) to smaller complexes (<60 kDa). The results are consistent with HSP20 expression and macromolecular structure being dynamically regulated in airway smooth muscle. HSP20 is upregulated by beta agonists and downregulated by proinflammatory cytokines. HSP20 is phosphorylated in vivo in a cAMP-dependent manner and the phosphorylated form promotes airway smooth muscle relaxation, possibly through depolymerization of F-actin as well as inhibition of myosin binding to actin.

Possible Mechanisms for the Effect of Protein Sensitization on Contractile Function of Fast and Slow Muscles in Mice

We studied the mechanisms underlying the effect of immunobiological reorganization of the organism on contractile function of isolated skeletal muscles from mouse leg (fast muscle, m. extensor digitorum longus; and slow muscle, m. soleus). Protein sensitization was accompanied by changes in contractile properties of fast and slow skeletal muscles. These changes were differently directed in muscles with various phenotypes. The force of carbachol-induced contraction (cholinergic agonist) increased in the slow muscle, but decreased in the fast muscle. The direction of changes in the force of carbachol-induced contractions under conditions of protein sensitization in skeletal muscles correlates with changes in non-quantal secretion of acetylcholine in the endplate (H-effect). Opposite changes in functional properties of fast and slow muscles from mouse leg during protein sensitization are related to choline-mediated excitation of the muscle fiber membrane. Our results suggest that changes in contractile function of skeletal muscles during protein sensitization are associated with variations in choline-mediated excitation of the muscle fiber membrane and modification of electromechanical coupling.

Expression in Pichia pastoris and Backbone Dynamics of Dendroaspin and Erabutoxin b: Motion Variability within Three‐fingered Toxins

Dendoaspin (DEN) and Erabutoxin b (EBTb) are three‐fingered toxins which possess a similar architecture with unrelated biological functions. They share a common structure consisting of a β‐sheet core that tightly cross‐linked by four conserved disulfide bonds. DEN inhibits platelet aggregation by blocking the binding of fibrinogen to the integrin αIIbβ3 of platelets. In contrast, EBTb is a potent inhibitor of nicotinic acetylcholine receptor. To study their function and dynamics relationships, we expressed DEN and EBTb in Pichia pastoris (P. pastoris). The recombinant DEN expressed in P. pastoris inhibited platelet aggregation with a KI of 149 nM. The recombinant EBTb expressed in P. pastoris inhibited carbachol‐induced muscle contraction with a KI of 42 nM. They are the same potency as those of native proteins. NMR analysis showed that DEN and EBTb expressed in P. pastoris processes the same structures as those of native proteins. The results provide the first direct evidence that highly disulfide‐bonded three‐fingered toxins can be expressed in P. pastoris with the correct fold. Analysis of the dynamics properties of DEN and EBTb showed that they exhibited flexibility on the loops II and III and motions on multiple time scales. The RGD‐containing loop of DEN was the most flexible region and the apex of the loops II and III of EBTb exhibited flexible motions on the ps/ns timescale. Comparison of the dynamics properties of DEN and EBTb with other members of three‐fingered toxins suggests that their dynamics deviations may play an important role in interacting with different target proteins.

Phospholipase C mediated Ca 2+ signals in murine urinary bladder smooth muscle

Muscarinic stimulation of urinary bladder induces contraction via an increase in intracellular Ca 2+ concentration that results from Ca 2+ influx through Ca 2+ channels and/or IP 3-mediated Ca 2+ release controlled by phospholipase C (PLC) signalling. The significance of PLC/IP 3 signalling in this cascade has recently been questioned because PLC inhibitors were without effect on carbachol-induced contractions in detrusor muscle strips. However, PLC/IP 3-mediated Ca 2+ release was clearly observed in recordings of Ca 2+ signals in isolated myocytes. Therefore, we investigated the presence of PLC/IP 3-dependent Ca 2+ release by directly monitoring Ca 2+ signals in intact detrusor muscle strips. Concomitant Ca 2+ signals from Ca 2+ channel activity were eliminated by the Ca 2+ channel antagonist isradipine (3 µM) or by the use of muscles from Ca v1.2 channel-deficient (SMACKO) mice. In absence of Ca 2+ channel activity, carbachol elicited contractions and Ca 2+ signals in muscles from wild type and SMACKO mice that were inhibited by the PLC inhibitor U73122 (10 µM). The results show that PLC/IP 3-dependent Ca 2+ release is activated by stimulation with carbachol in urinary bladder smooth muscle but has a minor contribution to overall carbachol-induced Ca 2+ signals.

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.

Alterations in bladder function associated with urothelial defects in uroplakin II and IIIa knockout mice

The effects of deleting genes encoding uroplakins II (UPII) and III (UPIIIa) on mouse bladder physiology/dysfunction were studied in male and female wild type and knockout (KO) mice. UPII, UPIIIa, and WT mice were catheterized using previously described techniques. Continuous cystometry was conducted in conscious, freely moving animals. Bladder strips were harvested after animal sacrifice and pharmacological studies and EFS were conducted in an organ chamber. Histological studies were also carried on with H&E staining to identify differences among the three mouse types. These studies have revealed numerous alterations, some of which were apparently gender-specific. Nonvoiding contractions were common in both UPII and UPIIIa KO mice, although more severe in the former. In particular, the increased bladder capacity, micturition pressure and demonstrable nonvoiding contractions observed in the male UPII KO's, were reminiscent of an obstruction-like syndrome accompanied by evidence of emerging bladder decompensation, as reflected by an increased residual volume. Pharmacological studies revealed a modest, gender-specific reduction in sensitivity of isolated detrusor strips from UPII KO female mice to carbachol-induced contractions. A similar reduction was observed in UPIIIa KO female mice. Histological investigation showed urothelial hyperplasia in both UPII KO and UPIIIa KO mice, although again, apparently more severe in the former. These results confirm and extend previous work to indicate that urothelial defects due to uroplakin deficiency are associated with significant alterations in bladder function and further highlight the importance of the urothelium to bladder physiology/dysfunction.

In vivo and in vitro pharmacological characterization of SVT‐40776, a novel M3 muscarinic receptor antagonist, for the treatment of overactive bladder

Highly selective M(3) muscarinic receptor antagonists may represent a better treatment for overactive bladder syndrome, diminishing side effects. Cardiac side effects of non-selective antimuscarinics have been associated with activity at M(2) receptors as these receptors are mainly responsible for muscarinic receptor-dependent bradycardia. We have investigated a novel antimuscarinic, SVT-40776, highly selective for M(3) over M(2) receptors (Ki = 0.19 nmol.L(-1) for M(3) receptor affinity). This study reports the functional activity of SVT-40776 in the bladder, relative to its activity in atria. In vitro and ex vivo (oral dosing) inhibition of mouse detrusor and atrial contractile responses to carbachol were used to study the functional activity of SVT-40776. The in vivo efficacy of SVT-40776 was characterized by suppression of isovolumetric spontaneous bladder contractions in anaesthetized guinea pigs after intravenous administration. SVT-40776 was the most potent in inhibiting carbachol-induced bladder contractions of the anti-cholinergic agents tested, without affecting atrial contractions over the same range of concentrations. SVT-40776 exhibited the highest urinary versus cardiac selectivity (199-fold). In the guinea pig in vivo model, SVT-40776 inhibited 25% of spontaneous bladder contractions at a very low dose (6.97 microg.kg(-1) i.v), without affecting arterial blood pressure. SVT-40776 is a potent inhibitor of M(3) receptor-related detrusor contractile activity. The absence of effects on isolated atria preparations represents an interesting characteristic and suggests that SVT-40776 may lack unwanted cardiac effects; a feature especially relevant in a compound intended to treat mainly elderly patients.

Alchornea Cordifolia (Euphorbiaceae), the Major Constituent of Antiasthmatic Herbal Formulations in Ghana, Stimulates β- adrenoceptors.

We have investigated the scientific basis for the traditional use of Alchornea cordifolia (Euphorbiaceae), by using isolated tissue models of smooth muscles in the laboratory. The aqueous extract of the leaves of Alchornea cordifolia produced a dose-related relaxation of the isolated rabbit duodenum in a manner similar to noradrenaline, adrenaline and isoprenaline, which were antagonised by propranolol (10μg/ml) by 63% but not phentolamine, suggesting a non-specific β- receptor-mediated activity. Additionally, the extract (12mg) evoked increased rate and force of contraction similar to isoprenaline on the Langendorff's isolated perfused rabbit heart preparation by 87% by an action on β1- receptors. These effects were blocked by propranolol (0.1mg), a nonspecific β-blocker. Carbachol-induced contractions on the guinea-pig tracheal smooth muscle were reversed by the extract in a cumulative dose manner, akin to the bronchodilator actions of isoprenaline. Similarly, 4μg/ml carbachol-elicited contractions on both pregnant and non-pregnant rat uterus were antagonised by the extract (50mg/ml) by 72% and 64% respectively. All together, our data suggest that the extract stimulates smooth muscles through activation of β-adrenoceptors in a manner similar to isoprenaline, which supports its traditional use as a constituent of anti-asthmatic and cough formulations, and in the control of spontaneous abortion. Journal of the Ghana Science Association Vol. 10 (2) 2008: pp. 1-11

Contribution of RhoA kinase and protein kinase C to weak relaxant effect of pinacidil on carbachol-induced contractions in sensitized guinea-pig trachealis

The exact mechanisms underlying the weak bronchodilator effect of K(ATP) channel openers on cholinergic stimulations is unknown. The present study was designed to examine the relaxant efect of pinacidil in guinea-pig trachea stimulated with carbachol by the presence of calcium sensitizer inhibitors; HA 1077, a rhoA kinase inhibitor, and chelerythrine, a protein kinase C inhibitor. Adenosine (10 microM) was used as other contractile agent for comparison. Tracheal tissues were isolated from ovalbumin sensitized guineapigs and changes in tension were recorded isometrically. Pinacidil (1-100 muM, cumulatively) and HA 1077 (0.01-30 microM, cumulatively) produced concentration-dependent relaxations in unstimulated tisues. The relaxant response to pinacidil decreased in carbachol contracted tissues, but increased in adenosine-stimulated tissues. Pretreatment of the tissues with HA 1077 (0.1 microM) and chelerythrine (10 microM) increased the pinacidil-induced relaxations by approximately %100 and %40, respectively. Glibenclamide, a KATP channel blocker, partially antagonized the pinacidil response in contracted tissues. Glibenclamide also inhibited the carbachol and adenosine induced contractions. These results suggest that diminish effect of pinacidil may have related to the enhanced calcium sensitization by cholinergic stimulation. Rho kinase inhibitors appear more effective than PKC inhibitors to achieve of this failure.

Potentiation of carbachol-induced detrusor smooth muscle contractions by β-adrenoceptor activation

In strips of rabbit bladder free of urothelium, the β-adrenoceptor agonist, isoproterenol, significantly reduced basal detrusor smooth muscle tone and inhibited contractions produced by low concentrations of the muscarinic receptor agonist, carbachol. During a carbachol concentration–response curve, instead of inhibiting, isoproterenol strengthened contractions produced by high carbachol concentrations. Thus, the carbachol concentration–response curve was shifted by isoproterenol from a shallow, graded relationship, to a steep, switch-like relationship. The tyrosine kinase inhibitor, genistein, inhibited carbachol-induced contractions only in the presence of isoproterenol. Contraction produced by a single high carbachol concentration (1 µM) displayed 1 fast and 1 slow peak. In the presence of isoproterenol, the slow peak was not strengthened, but was delayed, and U-0126 (mitogen-activated protein kinase kinase inhibitor) selectively inhibited this delay concomitantly with inhibition of extracellular signal-regulated kinase (ERK) phosphorylation. Isoproterenol reduced ERK phosphorylation only in the absence of carbachol. These data support the concept that, by inhibiting weak contractions, potentiating strong contractions, and producing a more switch-like concentration–response curve, β-adrenoceptor stimulation enhanced the effectiveness of muscarinic receptor-induced detrusor smooth muscle contraction. Moreover, β-adrenoceptor stimulation changed the cellular mechanism by which carbachol produced contraction. The potential significance of multi-receptor and multi-cell crosstalk is discussed.

Curcumin reverses attenuated carbachol-induced contraction of the colon in a rat model of colitis

Background- Curcumin ameliorates colitis whether it reverses colitis-induced reduction in colonic contractility remains to be investigated. Objectives- To investigate the effect of curcumin on colitis-induced reduction of carbachol-induced contraction in colon segments from rats treated with trinitrobenzenesulphonic acid. Methods- Colitis was induced in rats by intra rectal administration of trinitrobenzenesulphonic acid and followed for 5 days. A group of animals which received trinitobenzene sulphonic acids was treated with curcumin (100 mg/Kg and 200 mg/kg body weight) 2 hrs prior to induction of colitis. The controls received phosphate buffered saline in a similar fashion. Markers of inflammation and contractility of colon were assayed using standard procedures. Results- Induction of colitis was associated with increased myeloperoxidase activity and malondialdehyde levels, gross histological changes characterized by infiltration of inflammatory cells. All these changes were prevented by treatment with curcumin (100mg/kg). Treatment with curcumin also reduced the histological scores from 3.34±0.40 to 1.75±0.30 confirming an anti-inflammatory effect of curcumin in this experimental model of colitis. Colonic reactivity to carbachol was decreased in colitis affecting the maximum response but not sensitivity. Treatment with curcumin had no effect on sensitivity of the colon to carbachol in any of the preparations. Curcumin however reversed the decrease in carbachol-induced contraction associated with trinitrobenzenesulphonic acid treatment. The same dose of curcumin had no effect on either the potency of or the maximum response to carbachol in control rats. Tissue expression of NF-kB was increased in colon segments from trinitrobenzenesulphonic acid -treated rats and this was inhibited in rats treated with curcumin. Conclusions- Based on these findings it is concluded that curcumin prevented the reduction in carbachol-induced contraction in trinitrobenzenesulphonic acid -treated rats by modulating NF-kB signaling pathway.

17.BETA.-Estradiol Induces Gastrointestinal Motility Disorder by Decreasing CPI-17 Phosphorylation Via Changes in Rho-Family G-Protein Rnd Expression in Small Intestine

In the present study, we investigated the long-term effects of 17beta-estradiol on the motility of small intestine in in vitro organ culture and in vivo treatment studies. When rat ileal circular smooth muscle tissues were cultured with 17beta-estradiol (0.1 and 1 microM) for 5 days, carbachol-induced contraction was inhibited. In ileal tissue isolated from ovariectomized rat treated with 17beta-estradiol (200 microg/kg/day s.c. for 3 days), carbachol-induced contraction was also impaired. Both in vitro and in vivo, 17beta-estradiol treatment upregulated heat shock protein 27 (HSP27) expression, indicating the activation of estrogen receptor in the intestinal smooth muscle. 17beta-estradiol did not change protein expression levels of RhoA and RhoA-associated coiled coil-forming serine/threonine kinases (ROCKs); however, it upregulated Rnd2 and Rnd3, Rho-family G-proteins that counteract the functions of RhoA, both in vitro and in vivo. In organ culture, treatment of ileal tissue with 17beta-estradiol greatly suppressed the carbachol-induced increase in phosphorylation at Thr38 in CPI-17 without altering total CPI-17 protein expression. These results suggest that 17beta-estradiol upregulates Rnd expression to inhibit the RhoA-mediated Ca(2+) sensitization of contractile mechanisms, which are mediated by CPI-17 phosphorylation in ileal smooth muscle. This mechanism may contribute to the intestinal motility disorder occurring in gender-dependent bowel diseases.

Inhibitory effect of quercetin on rat trachea contractility &lt;I&gt;in vitro&lt;/I&gt;

The effect of quercetin, a naturally occurring flavonoid traditionally used to treat airway diseases such as bronchial asthma, on the contractile response elicited by electrical field stimulation or carbachol in rat isolated trachea was investigated. Isolated tracheal tissue was subjected to contractions by an electrical field stimulation of 5 Hz for 30 s, 400 mA, and the responses in the presence of cumulative concentrations of quercetin (10(-6)-3x10(-4) M) were observed. The effect of quercetin was also evaluated after administration of phentolamine plus propranolol (to block alpha- and beta-adrenergic receptors), NG-nitro-L-arginine methyl ester (to block nitric oxide synthesis), capsaicin (to desensitise sensory C fibres), alpha-chymotrypsin (a proteolytic enzyme that rapidly degrades vasoactive intestinal peptide), SR140333 and SR48968 (tackykinin NK1 and NK2 receptor antagonists, respectively). Quercetin produced a concentration-dependent inhibition of contractions induced by both carbachol and electrical field stimulation. However, quercetin was more active in inhibiting the contractions produced by electrical field stimulation than those induced by carbachol, suggesting a presynaptic site of action (in addition to a postsynaptic effect, as revealed by the inhibitory action of quercetin on carbachol-induced contractions). The inhibitory effect of quercetin on contractions induced by electrical field stimulation was unaffected by phentolamine plus propranolol, SR 140333 and SR 48968, capsaicin treatment or by the proteolytic enzyme alpha-chymotrypsin. In contrast, the nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester significantly reduced the inhibitory effect of quercetin on contractions induced by electrical field stimulation. Quercetin inhibits rat tracheal contractility through a presynaptic (involving nitric oxide) and a postsynaptic site of action.

Carbachol Induces Ca2+-Dependent Contraction via Muscarinic M2 and M3 Receptors in Rat Intestinal Subepithelial Myofibroblasts

Intestinal myofibroblasts (IMFs) that exist adjacent to the basement membrane of intestines have contractility and contribute to physical barriers of the intestine. Nerve endings distribute adjacent to IMFs, suggesting neurotransmitters may influence IMFs motility; however, there is no direct evidence showing the interaction. Here, we isolated IMFs from rat colon and investigated the effect of acetylcholine on IMFs contractility. In the collagen gel contraction assay, carbachol (1 - 10 microM) and the muscarinic receptor agonist bethanechol (30 - 300 microM) dose-dependently induced IMFs contraction. Pretreatment with the muscarinic receptor antagonist atropine (1 - 10 nM) inhibited carbachol-induced contraction. In RT-PCR, mRNA expression of all muscarinic receptor subtypes (M(1) - M(5)) was detected in IMFs. Subsequently we found pretreatment with the muscarinic M(2) receptor antagonist 11-([2-[(diethylamino)methyl]-1-piperdinyl]acetyl)-5,11-dihydro-6H-pyrido[2,3-b][1,4]benzodiazepine-6-one (AF-DX116) (10 and 30 nM) or the muscarinic M(3) receptor antagonist 4-diphenylacetoxy-N-methyl-piperidine (4-DAMP) (3 and 10 nM) dose-dependently inhibited carbachol-induced contraction. In Ca(2+) measurement, 1 - 10 microM carbachol and 30 - 300 microM bethanechol elevated the intracellular Ca(2+) concentration ([Ca(2+)](i)) in IMFs. Atropine (10 nM) eliminated carbachol-induced [Ca(2+)](i) elevation. The Ca(2+)-channel blocker LaCl(3) (3 microM) abolished carbachol-induced [Ca(2+)](i) elevation and contraction. Furthermore, AF-DX116 and 4-DAMP dose-dependently inhibited the carbachol-induced [Ca(2+)](i) elevation. These observations suggest that acetylcholine elicits Ca(2+)-dependent IMF contraction through muscarinic M(2) and M(3) receptors.

Signal transduction pathways of muscarinic receptor mediated activation in the newborn and adult mouse urinary bladder

To study the role of M(2) and M(3) muscarinic receptor subtypes, sources of activator Ca(2+), and mechanisms involved in increased force oscillations in muscarinic contractions in the bladders of newborn and adult mice, as in the adult bladder muscarinic M(3) receptors are considered to mediate the main part of bladder contraction, and this has not been established in the newborn bladder. Bladder preparations from newborn (0-2 days) and adult (10-12 weeks) mice were mounted for in vitro force registration and activated with carbachol and high-K(+) solution in the presence of M(3) (4-DAMP 30 nM) or M(2) (methoctramine, 100 nM) receptor antagonists. Thapsigargin (1 microm) or ryanodine (10 microm) were used to inhibit sarcoplasmic reticulum Ca(2+) release. L-NAME (300 microm) and 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 10 microm) were used to inhibit nitric oxide synthase and guanylyl cyclase, respectively. Gap-junction function was inhibited with by 18-beta-glycyrrhetinic acid (18-beta-GA; 0.1-100 microm). Big-conductance (BK) and small-conductance (SK) K(+) channels were inhibited by apamine and charybdotoxin (0.3 microm), respectively. Concentration-response relations for carbachol in the presence of 4-DAMP and methoctramine showed that M(3) receptors are the main activating pathway also in the newborn bladder. Neither thapsigargin nor ryanodine influenced the muscarinic responses of the newborn and adult bladders. Carbachol-induced contractions were not influenced by L-NAME or ODQ. The 18-beta-GA inhibited carbachol-induced contractions in both newborn and adult tissue in a similar manner. Apamine and charybdotoxin slightly increased the amplitude of the contractile responses. These results suggest that in the newborn mouse bladder, as in adult bladders, the M(3) muscarinic receptor subtype is mainly responsible for carbachol-induced contractile responses. The main mechanism for muscarinic receptor-induced activation is influx of Ca(2+) from the extracellular medium, and there seems to be no major contribution of Ca(2+) release from intracellular stores. The phasic contractile activity induced by carbachol in the newborn bladder is not influenced by gap junction inhibition and does not involve SK and BK channels.