Mechanisms underlying the nitric oxide inhibitory effects in mouse ileal longitudinal muscle

We investigated the mechanisms involved in the nitric oxide (NO)-induced inhibitory effects on longitudinal smooth muscle of mouse ileum, using organ bath technique. Exogenously applied NO, delivered as sodium nitroprusside (SNP; 0.1-100 micromol/L) induced a concentration-dependent reduction of the ileal spontaneous contractions. 1H-[1,2,4]oxadiazolol[4,3,a]quinoxalin-1-one (ODQ; 1 micromol/L), a guanilyl cyclase inhibitor, reduced the SNP-induced effects. Tetraethylammonium chloride (20 mmol/L), a non-selective K+ channel blocker, and charybdotoxin (0.1 micromol/L), blocker of large conductance Ca2+-dependent K+ channels, significantly reduced SNP-induced inhibitory effects. In contrast, apamin (0.1 micromol/L), blocker of small conductance Ca2+-dependent K+ channels, was not able to affect the response to SNP. Ciclopiazonic acid (10 micromol/L) or thapsigargin (0.1 micromol/L), sarcoplasmatic reticulum Ca2+-ATPase inhibitors, decreased the SNP-inhibitory effects. Ryanodine (10 micromol/L), inhibitor of Ca2+ release from ryanodine-sensitive intracellular stores, significantly reduced the SNP inhibitory effects. The membrane permeable analogue of cGMP, 8-bromoguanosine 3',5'-cyclic monophosphate (100 micromol/L), also reduced spontaneous mechanical activity, and its effect was antagonized by ryanodine. The present study suggests that NO causes inhibitory effects on longitudinal smooth muscle of mouse ileum through cGMP which in turn would activate the large conductance Ca2+-dependent K+ channels, via localized ryanodine-sensitive Ca2+ release.     

A1 receptors mediate adenosine inhibitory effects in mouse ileum via activation of potassium channels

AimsWe investigated the effects induced by exogenous adenosine on the spontaneous contractile activity of the longitudinal muscle of a mouse ileum, the receptor subtypes activated, the involvement of enteric nerves and whether opening of K⁺ channels was a downstream event leading to the observed effects.Main methodsMechanical responses of the mouse ileal longitudinal muscle to adenosine were examined in vitro as changes in isometric tension.Key findingsAdenosine caused a concentration-dependent reduction of the spontaneous contraction amplitude of the ileal longitudinal muscle up to its complete disappearance. This effect induced was markedly reduced by an A₁ receptor antagonist, but not by A₂ and A₃ receptor antagonists and mimicked only by the A₁ receptor agonist. Adenosine uptake inhibitors did not change adenosine potency. A₁ receptor expression was detected at the smooth muscle level. Adenosine responses were insensitive to tetrodotoxin, atropine or nitric oxide synthase inhibitor. Tetraethylammonium and iberiotoxin, BK_Ca channel blockers, significantly reduced adenosine effects, whilst 4-aminopyridine, a K_v blocker, apamin, a small conductance Ca²⁺-activated K⁺ (SK_Ca) channel blocker, charybdotoxin, an intermediate conductance Ca²⁺-activated K⁺ (IK_Ca) and BK_Ca channel blocker, or glibenclamide, an ATP-sensitive K⁺ channel blocker, had no effects. The combination of apamin plus iberiotoxin caused a reduction of the purinergic effects greater than iberiotoxin alone.SignificanceAdenosine acts as an inhibitory modulator of the contractility of mouse ileal longitudinal muscle through postjunctional A₁ receptors, which in turn would induce opening of BK_Ca and SK_Ca potassium channels. This study would provide new insight in the pharmacology of purinergic receptors involved in the modulation of the gastrointestinal contractility.     

Phasic Contractions of the Mouse Vagina and Cervix at Different Phases of the Estrus Cycle and during Late Pregnancy

Background/Aims

The pacemaker mechanisms activating phasic contractions of vaginal and cervical smooth muscle remain poorly understood. Here, we investigate properties of pacemaking in vaginal and cervical tissues by determining whether: 1) functional pacemaking is dependent on the phase of the estrus cycle or pregnancy; 2) pacemaking involves Ca²⁺ release from sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPase (SERCA) -dependent intracellular Ca²⁺ stores; and 3) c-Kit and/or vimentin immunoreactive ICs have a role in pacemaking.

Methodology/Principal Findings

Vaginal and cervical contractions were measured in vitro, as was the distribution of c-Kit and vimentin positive interstitial cells (ICs). Cervical smooth muscle was spontaneously active in estrus and metestrus but quiescent during proestrus and diestrus. Vaginal smooth muscle was normally quiescent but exhibited phasic contractions in the presence of oxytocin or the K⁺ channel blocker tetraethylammonium (TEA) chloride. Spontaneous contractions in the cervix and TEA-induced phasic contractions in the vagina persisted in the presence of cyclopiazonic acid (CPA), a blocker of the SERCA that refills intracellular SR Ca²⁺ stores, but were inhibited in low Ca²⁺ solution or in the presence of nifedipine, an inhibitor of L-type Ca²⁺channels. ICs were found in small numbers in the mouse cervix but not in the vagina.

Conclusions/Significance

Cervical smooth muscle strips taken from mice in estrus, metestrus or late pregnancy were generally spontaneously active. Vaginal smooth muscle strips were normally quiescent but could be induced to exhibit phasic contractions independent on phase of the estrus cycle or late pregnancy. Spontaneous cervical or TEA-induced vaginal phasic contractions were not mediated by ICs or intracellular Ca²⁺ stores. Given that vaginal smooth muscle is normally quiescent then it is likely that increases in hormones such as oxytocin, as might occur through sexual stimulation, enhance the effectiveness of such pacemaking until phasic contractile activity emerges.     

Mechanisms Underlying Nitroglycerin-Induced Suppression of Phenylephrine- and Caffeine-Evoked Contractions of Smooth Muscles of the Rabbit Main Pulmonary Artery

Using a strain measurement technique, we studied the mechanisms of the effect of a nitric oxide (NO) donor, nitroglycerin (NG), on contractions of smooth muscles of the main pulmonary artery of the rabbit induced by phenylephrine and caffeine in normal Krebs solution (NKS) or in nominally calcium-free solution (NCFS). Phenylephrine applications caused contractions consisting of an initial fast phasic low-amplitude component followed by a tonic higher-amplitude component. After caffeine-induced monophasic low-amplitude contraction, tension of the smooth muscle strip shifted below the conventional zero. Addition of NG to NKS resulted in a decrease in the smooth muscle tension below the conventional zero. Under the influence of NG, the initial phasic component of phenylephrine-induced contraction was partially suppressed, whereas the next tonic component was suppressed to a greater extent. At the same time, NG exerted nearly no influence on the amplitude of caffeine-induced contractions. Washing out by NKS of phenylephrine dissolved in NCFS resulted in initiation of a fast phasic high-amplitude contraction. Such a contraction did not develop either in the presence of NG or phenylephrine in NCFS or in the case of washing out of caffeine dissolved in NCFS. Our findings allow us to conclude that phenylephrine or caffeine added to the superfusate induce contractions of the smooth muscle cells (SMC) of the main pulmonary artery of the rabbit due to activation of Ca²⁺ release from the respective intracellular calcium stores. In addition, calcium ions entering SMC through the calcium channels of the plasma membrane are also involved in activation of the phenylephrine-induced contraction. The inhibitory effect of NG on the phenylephrine-induced contraction is related to the influence of NO on the release of Ca²⁺ from the inositol trisphosphate-sensitive intracellular calcium store and receptor-operated inflow of Ca²⁺ to SMC. Nitroglycerin did not significantly influence the caffeine-induced contraction and, therefore, Ca²⁺ release from the caffeine-sensitive store.     

Accelerated communication the direct contractile effect of gastrin releasing peptide on isolated gastric smooth muscle cells of the guinea pig

Smooth muscle cells isolated from the gastric muscle layers of the guinea pig were used to determine whether gastrin releasing peptide (GRP) can cause contraction by exerting a direct action on muscle cells. In addition, the inhibitory effect of 8-( N,N-diethylamino )-octyl-3,4,5-trimethoxybenzoate hydrochloride ( TMB-8 ), an inhibitor of intracellular Ca²⁺ release, and verapamil, a Ca²⁺ channel blocker, on the GRP-induced contraction of gastric smooth muscle cells were examined. GRP elicited a contractile response of gastric muscle cells in a dose-dependent manner. The ED₅₀ was 13 pM. TMB-8 significantly inhibited the contractile effect of GRP in gastric muscle cells. These results demonstrate the direct action of GRP on the gastric smooth muscle cells of the guinea pig, and the importance of Ca²⁺-release from intracellular calcium stores in the contractile response to GRP.     

Aberrant Ca2+ oscillations in smooth muscle cells from overactive human bladders

Overactive bladder (OAB) syndrome is highly prevalent and costly, but its pathogenesis remains unclear; in particular, the origin of involuntary detrusor muscle activity. To identify the functional substrate for detrusor muscle overactivity, we examined intracellular Ca²⁺ oscillations in smooth muscle cells from pathologically overactive human bladders. Basal cytoplasmic Ca²⁺ concentration was elevated in smooth muscle cells from overactive bladders. Unprovoked, spontaneous rises of Ca²⁺ were also identified. These spontaneous Ca²⁺ oscillations were Ca²⁺-dependent, sensitive to L-type Ca²⁺ channel antagonist verapamil and also attenuated by blocking SR Ca²⁺ reuptake. The fraction of spontaneously active cells was higher in cells from overactive bladders and the magnitude of spontaneous Ca²⁺ oscillations also greater. Spontaneous action potentials or depolarising oscillations were also observed, associated with Ca²⁺ rise; with a higher percentage of cells from idiopathic OAB, but not in neurogenic OAB. Low concentrations of NiCl₂ attenuated both spontaneous electrical and Ca²⁺ activation. This study provides the first evidence that spontaneous, autonomous cellular activity—Ca²⁺ and membrane potential oscillations, originates from detrusor smooth muscle in human bladders, mediated by extracellular Ca²⁺ influx and intracellular release. Such cellular activity underlies spontaneous muscle contraction and defective Ca²⁺ activation contributes to up-regulated contractile activity in overactive bladders.     

The role of TRPP2 in agonist-induced gallbladder smooth muscle contraction

TRPP2 channel protein belongs to the superfamily of transient receptor potential(TRP) channels and is widely expressed in various tissues, including smooth muscle in digestive gut. Accumulating evidence has demonstrated that TRPP2 can mediate Ca~(2+) release from Ca~(2+) stores. However, the functional role of TRPP2 in gallbladder smooth muscle contraction still remains unclear. In this study, we used Ca~(2+) imaging and tension measurements to test agonist-induced intracellular Ca~(2+) concentration increase and smooth muscle contraction of guinea pig gallbladder, respectively. When TRPP2 protein was knocked down in gallbladder muscle strips from guinea pig, carbachol(CCh)-evoked Ca~(2+) release and extracellular Ca~(2+) influx were reduced significantly, and gallbladder contractions induced by endothelin 1 and cholecystokinin were suppressed markedly as well. CCh-induced gallbladder contraction was markedly suppressed by pretreatment with U73122, which inhibits phospholipase C to terminate inositol 1,4,5-trisphosphate receptor(IP3) production, and 2-aminoethoxydiphenyl borate(2APB), which inhibits IP3 recepor(IP3R) to abolish IP3R-mediated Ca~(2+) release. To confirm the role of Ca~(2+) release in CCh-induced gallbladder contraction, we used thapsigargin(TG)-to deplete Ca~(2+) stores via inhibiting sarco/endoplasmic reticulum Ca~(2+)-ATPase and eliminate the role of store-operated Ca~(2+) entry on the CCh-induced gallbladder contraction. Preincubation with 2 μmol L~(-1) TG significantly decreased the CCh-induced gallbladder contraction. In addition, pretreatments with U73122, 2APB or TG abolished the difference of the CCh-induced gallbladder contraction between TRPP2 knockdown and control groups. We conclude that TRPP2 mediates Ca~(2+) release from intracellular Ca~(2+) stores, and has an essential role in agonist-induced gallbladder muscle contraction.     

Endothelin contraction in pig coronary: Receptor types and Ca2+-mobilization

Endothelin is one of the most potent vasoconstrictors known. It plays an important role in the regulation of vascular tone and in the development of many cardiovascular diseases. This study focuses on the receptor types and the Ca²⁺ mobilization responsible for endothelin-1 (ET-1) contraction in de-endothelialized pig coronary artery rings. ET-1 contracted the artery rings with an EC₅₀ = 6.5 ± 1 nM and a maximum contraction which was 98.6 ± 9% of the contraction produced by 60 mM KCl. BQ123 (5 µM), an ET_A antagonist, reversed 78 ± 3% of the ET-1 contraction (50 nM). IRL1620, a selective ET_B agonist, produced 23 ± 3% of the total ET-1 contraction with an EC₅₀ = 12.7 ± 2 nM. More than 85% of the contraction due to 100 nM IRL 1620 was inhibited by 200 nMBQ788, an ET_B antagonist. Therefore, approximately 80% of the ET-1 contraction in this artery occurred via ET_A receptors, and the other 20% was mediated by ET_B receptors. To assess the Ca²⁺ pools utilized during the ET-1 response, ET-1 contraction was also examined in medium containing an L-type Ca²⁺ channel blocker nitrendipine, and in Ca²⁺ free medium containing 0.2 mM EGTA. In Ca²⁺ containing medium the contraction elicited by ET-1 was 98.6 ± 9% of the KCl contraction, however, in the presence 10 µM nitrendipine the ET-1 induced contraction was 54 ± 7% of the KCl contraction, and in Ca²⁺-free medium it was 13 ± 2%. Similarly, the IRL 1620 contractions in Ca²⁺ containing medium, in the presence of nitrendipine and in Ca²⁺-free medium were 22.4 ± 3%, 12 ± 3% and 11 ± 2% of the KCl response respectively. Thus, both ET_A and ET_B contractions utilize extracellular Ca²⁺ pools via L-type Ca²⁺ channels and other undefined route(s), as well as intracellular Ca²⁺ pools. In the pig coronary artery smooth muscle, ET-1 contractions occur predominantly via ET_A receptors, with ET_B receptors using similar Ca²⁺ mobilization pathways, but the ET_B receptors appear to use the intracellular Ca²⁺ stores to a greater extent.     

Optical induction of muscle contraction at the tissue scale through intrinsic cellular amplifiers

The smooth muscle cell is the principal component responsible for involuntary control of visceral organs, including vascular tonicity, secretion, and sphincter regulation. It is known that the neurotransmitters released from nerve endings increase the intracellular Ca²⁺ level in smooth muscle cells followed by muscle contraction. We herein report that femtosecond laser pulses focused on the diffraction‐limited volume can induce intracellular Ca²⁺ increases in the irradiated smooth muscle cell without neurotransmitters, and locally increased intracellular Ca²⁺ levels are amplified by calcium‐induced calcium‐releasing mechanisms through the ryanodine receptor, a Ca²⁺ channel of the endoplasmic reticulum. The laser‐induced Ca²⁺ increases propagate to adjacent cells through gap junctions. Thus, ultrashort‐pulsed lasers can induce smooth muscle contraction by controlling Ca²⁺, even with optical stimulation of the diffraction‐limited volume. This optical method, which leads to reversible and reproducible muscle contraction, can be used in research into muscle dynamics, neuromuscular disease treatment, and nanorobot control. (© 2014 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

The role of Ca2+ in the contractility of rabbit small intestine in vitro.

This study evaluated the role of Ca2+ in spontaneous and ACh- and KCl-induced contractions in longitudinal and circular smooth muscle from rabbit small intestine in vitro. In the first experiment, the amplitude, frequency and tone of spontaneous contractions in longitudinal and circular smooth muscle of small intestine were determined and, in the second experiment, the ACh- and KCl-induced responses of longitudinal and circular smooth muscle were measured. Atropine and guanethidine reduced the amplitude and tone of contractions in longitudinal and circular muscle, but reduced the frequency of contractions in circular muscle, only. TTX attenuated the amplitude of contractions and decreased the tone of contractions in longitudinal muscle, but increased the tone in circular muscle. Ca2+-free solutions, verapamil, nifedipine and caffeine diminished the three parameters of spontaneous contractions. Thapsigargin and cyclopiazonic acid increased the amplitude and tone of contractions in ileum longitudinal muscle, only, and cyclopiazonic acid increased the amplitude of contractions in circular muscle. Ca2+-free solutions, verapamil, nifedipine, thapsigargin, cyclopiazonic acid, and caffeine diminished ACh- and KCl-induced contractions. Those results suggest that extracellular Ca2+ plays a role in spontaneous contractions, and extracellular and intracellular Ca2+ participate in the ACh- and KCl-induced contractions of rabbit small intestine.     

The RyR–ClCa–VDCC axis contributes to spontaneous tone in urethral smooth muscle

Current therapies including pharmaceutical intervention and surgery have limited efficacy on stress urinary incontinence (SUI). One type of SUI is due to low intraurethral pressure caused by the disabled contraction of urethral smooth muscle (USM). However, the molecular mechanisms underlying the motility of USM remain unknown. Here, we show that USM represents spontaneous tone after stretching in humans and mice. Deletion of TMEM16A in the smooth muscle of mice abolishes spontaneous urethral tone. Furthermore, Cl_Ca currents and [Ca²⁺]_i in TMEM16A^SMKO mice were largely impaired. Inhibitors of ryanodine receptor (RyR), TMEM16A encoded calcium-activated chloride channel (Cl_Ca) and L-type voltage-dependent calcium channel (VDCC) fully prevented spontaneous tone accompanied by a significant decrease of intracellular calcium concentration ([Ca²⁺]_i). In summary, RyR–Cl_Ca–VDCC signaling contributes to spontaneous USM tone. This finding may provide a new promising approach for women with stress SUI who reject surgery.     

Ca2+ release and contraction induced by IP3 and contractile agonists in mammalian gastric smooth muscle

The source, time course and stoichiometry of cytosolic free Ca²⁺ ([Ca²⁺]_i) during contraction were examined in smooth muscle cells isolated from the guinea pig and human stomach. Contraction by receptor-linked agonists (eg, acetylcholine, cholecystokinin octapeptide and Met-enkephalin) was preceded by stoichiometric increases in [Ca²⁺]_i and net ⁴⁵Ca²⁺ efflux that were maintained in the absence of extracellular Ca²⁺ or in the presence of a Ca²⁺ channel blocker (13600). The intracellular Ca²⁺ store could be depleted by repeated stimulation with all agonists in Ca²⁺-free medium or in the presence of 13600 resulting in loss of contractile response; response was restored by re-exposure of the cells to Ca²⁺.The source of intracellular Ca²⁺ an the signal for its release were examined in saponin-permeabilized muscle cells. The cells retained their ability to contract in response to receptor-linked agonists and developed an ability to contract in response to inositol trisphosphate (IP₃). The cells accumulated Ca²⁺ to the same extent as intact muscle cells, but only in the presence of ATP. IP₃ caused a prompt, concentration-dependent increase in contraction, [Ca²⁺]_i and net ⁴⁵Ca²⁺ efflux. These effects were maximally similar to those produced by CCK-8 alone or in combination with IP₃: Depletion of the Ca²⁺ store by repeated stimulation of single muscle cells in Ca²⁺-free medium with IP₃, acetylcholine or CCK-8 separately resulted in loss of contractile response to all three agents; the response was restored by re-exposure of the muscle cell to a cytosol-like perfusate (Ca²⁺ 180 nM).The studies demonstrate that a product of membrane phosphoinositide hydrolysis is capable of mobilizing Ca²⁺ from a depletable, non-mitochondrial intracellular store that is utilized by receptor-linked agonists. The magnitude of IP₃-induced Ca²⁺ release is correlated with contraction.     

TPC2 Proteins Mediate Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP)- and Agonist-evoked Contractions of Smooth Muscle

Agonists such as those acting at muscarinic receptors are thought to induce contraction of smooth muscle primarily through inositol 1,4,5-trisphosphate production and release of Ca²⁺ from sarcoplasmic reticulum. However, the additional Ca²⁺-mobilizing messengers cyclic adenosine diphosphate ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP) may also be involved in this process, the former acting on the sarcoplasmic reticulum, the latter acting on lysosome-related organelles. In this study, we provide the first systematic analysis of the capacity of inositol 1,4,5-trisphosphate, cADPR, and NAADP to cause contraction in smooth muscle. Using permeabilized guinea pig detrusor and taenia caecum, we show that all three Ca²⁺-mobilizing messengers cause contractions in both types of smooth muscle. We demonstrate that cADPR and NAADP play differential roles in mediating contraction in response to muscarinic receptor activation, with a sizeable role for NAADP and acidic calcium stores in detrusor muscle but not in taenia caecum, underscoring the heterogeneity of smooth muscle signal transduction systems. Two-pore channel proteins (TPCs) have recently been shown to be key components of the NAADP receptor. We show that contractile responses to NAADP were completely abolished, and agonist-evoked contractions were reduced and now became independent of acidic calcium stores in Tpcn2^−/− mouse detrusor smooth muscle. Our findings provide the first evidence that TPC proteins mediate a key NAADP-regulated tissue response brought about by agonist activation of a cell surface receptor.     

Dissecting out the Complex Ca2+-Mediated Phenylephrine-Induced Contractions of Mouse Aortic Segments

L-type Ca²⁺ channel (VGCC) mediated Ca²⁺ influx in vascular smooth muscle cells (VSMC) contributes to the functional properties of large arteries in arterial stiffening and central blood pressure regulation. How this influx relates to steady-state contractions elicited by α1-adrenoreceptor stimulation and how it is modulated by small variations in resting membrane potential (V_m) of VSMC is not clear yet. Here, we show that α1-adrenoreceptor stimulation of aortic segments of C57Bl6 mice with phenylephrine (PE) causes phasic and tonic contractions. By studying the relationship between Ca²⁺ mobilisation and isometric tension, it was found that the phasic contraction was due to intracellular Ca²⁺ release and the tonic contraction determined by Ca²⁺ influx. The latter component involves both Ca²⁺ influx via VGCC and via non-selective cation channels (NSCC). Influx via VGCC occurs only within the window voltage range of the channel. Modulation of this window Ca²⁺ influx by small variations of the VSMC V_m causes substantial effects on the contractile performance of aortic segments. The relative contribution of VGCC and NSCC to the contraction by α1-adrenoceptor stimulation could be manipulated by increasing intracellular Ca²⁺ release from non-contractile sarcoplasmic reticulum Ca²⁺ stores. Results of this study point to a complex interactions between α1-adrenoceptor-mediated VSMC contractile performance and Ca²⁺ release form contractile or non-contractile Ca²⁺ stores with concomitant Ca²⁺ influx. Given the importance of VGCC and their blockers in arterial stiffening and hypertension, they further point toward an additional role of NSCC (and NSCC blockers) herein.     

A non-invasive vibrating calcium-selective electrode measures acetylcholine-induced calcium flux across the sarcolemma of a smooth muscle

To determine possible sources of Ca²⁺ during excitation-contraction coupling in smooth muscle, a vibrating Ca²⁺-selective electrode was used to measure Ca²⁺ flux during the process of contraction. The smooth muscle model was the longitudinal muscle of the body wall of a sea cucumberSclerodactyla briareus. Because acetylcholine caused slow contractions of the muscle that were inhibited by Ca²⁺ channel blockers diltiazem and verapamil in earlier mechanical studies, we chose a vibrating Ca²⁺-selective electrode as our method to test the hypothesis that acetylcholine may be stimulating Ca²⁺ influx across the sarcolemma, providing a Ca²⁺ source during excitation-contraction coupling. Acetylcholine treatment stimulated a net Ca²⁺ efflux that was both dose and time dependent. We then tested two L-type Ca²⁺ channel blockers, diltiazem and verapamil, and two non-specific Ca²⁺ blockers, cobalt (Co²⁺) and lanthanum (La³⁺) on acetylcholine-induced Ca²⁺ flux. All four Ca²⁺ blockers tested potently inhibited Ca²⁺ efflux induced by physiological doses of acetylcholine. We propose that the acetylcholine-induced Ca²⁺ efflux was the result of, first, Ca²⁺ influx through voltage-sensitive L-type Ca²⁺ channels, then the rapid extrusion of Ca²⁺ by an outwardly directed carrier such as the Na–Ca exchanger as suggested by Li⁺ substitution experiments. The vibrating Ca²⁺ electrode has provided new insights on the active and complex role the sarcolemma plays in Ca²⁺ homeostasis and regulating Ca²⁺ redistribution during excitation-contraction coupling.Abbreviations ACh acetylcholine - E-C coupling excitation-contraction coupling - LMBW longitudinal muscle of the body wall     

The effect of dietary supplementation of nitric oxide donor and inhibitor on nNOS expression in and motility of the small intestine of broilers

Abstract

We investigated the effect of dietary supplementation of sodium nitroprusside (SNP), a nitric oxide (NO) donor, and N-nitro-L-arginine methyl ester (L-NAME), a NO inhibitor, on neuronal nitric oxide synthase (nNOS) expression in and motility of small intestinum in broilers. A total of 560, one-day-old Ross 308 hybrid mixed sex broiler chicks were divided randomly into one control and seven treatment groups for a 42 day feeding trial including starter phase (0–21 days) and grower phase (22–42 days). The control group was fed a basal diet and the experimental groups were the fed basal diet supplemented with 25, 50, 100 and 200 mg/kg SNP and 25, 50 or 100 mg/kg L-NAME. Ten chickens from each group were sacrificed to collect samples on days 21 and 42. The expression patterns of nNOS immunoreactivity in nerve fibers were determined by immunohistochemistry. In the contractility studies, longitudinal isolated strips of duodenum, jejunum and ileum were treated with 10^?5 M L-arginine and 10^?4 M SNP. Immunohistochemistry revealed that nNOS expression was not detectable in the duodenum or ileum of either the control or experimental groups. On the other hand, nNOS immunoreactivity in the jejunum control group showed a strong reaction on day 21, but the reaction was weak on day 42. nNOS expression clearly was suppressed on day 21 by the diet supplemented with L-NAME, while the diet supplemented with SNP stimulated nNOS expression on day 21. Contractility experiments revealed that spontaneous contractility of isolated strips of duodenum, jejunum and ileum showed no significant difference among groups. Spontaneous contractions of all strips were inhibited by L-arginine and SNP in all groups. The percentage inhibition rate of spontaneous contractions of jejunum application on days 21 and 42 after L-arginine decreased in the group supplemented with 100 mg/kg L-NAME. The percentage inhibition rate on day 21 after SNP application decreased in both groups that received 50 and 100 mg/kg L-NAME. We demonstrated the expression pattern of nNOS in nerve fibers in jejunum of broiler chickens. Contractility studies revealed that the NOS-NO pathway may play a role in smooth muscle contraction of small intestine of chickens. Feeding strategies that supplement NO donor and NO inhibitor can be of physiological importance to small intestine motility owing to alteration of nNOS expression in the jejunum.     

Magnolol inhibits colonic motility through down-regulation of voltage-sensitive L-type Ca2+ channels of colonic smooth muscle cells in rats

This study aimed to investigate the effect of magnolol (5,5′-diallyl-2,2′-biphenyldiol) on contraction in distal colonic segments of rats and the underlying mechanisms. Colonic segments were mounted in organ baths for isometric force measurement. Whole-cell voltage-sensitive L-type Ca²⁺ currents were recorded on isolated single colonic smooth muscle cells using patch-clamp technique. The spontaneous contractions and acetylcholine (ACh)- and Bay K 8644-induced contractions were inhibited by magnolol (3–100 μM). In the presence of Bay K8644 (100 nM), magnolol (10–100 μM) inhibited the contraction induced by 10 μM ACh. By contrast, tetrodotoxin (100 nM) and N_ώ-nitro-l-arginine methyl ester (l-NAME 100 μM) did not change the inhibitory effect of magnolol (10 μM). In addition, magnolol (3–100 μM) inhibited the L-type Ca²⁺ currents. The present results suggest that magnolol inhibits colonic smooth muscle contraction through downregulating L-type Ca²⁺ channel activity.     

Involvement of Large-Conductance Ca2+-Activated K+ Channels in Chloroquine-Induced Force Alterations in Pre-Contracted Airway Smooth Muscle

The participation of large-conductance Ca²⁺ activated K⁺ channels (BKs) in chloroquine (chloro)-induced relaxation of precontracted airway smooth muscle (ASM) is currently undefined. In this study we found that iberiotoxin (IbTx, a selective inhibitor of BKs) and chloro both completely blocked spontaneous transient outward currents (STOCs) in single mouse tracheal smooth muscle cells, which suggests that chloro might block BKs. We further found that chloro inhibited Ca²⁺ sparks and caffeine-induced global Ca²⁺ increases. Moreover, chloro can directly block single BK currents completely from the intracellular side and partially from the extracellular side. All these data indicate that the chloro-induced inhibition of STOCs is due to the blockade of chloro on both BKs and ryanodine receptors (RyRs). We also found that low concentrations of chloro resulted in additional contractions in tracheal rings that were precontracted by acetylcholine (ACH). Increases in chloro concentration reversed the contractile actions to relaxations. In the presence of IbTx or paxilline (pax), BK blockers, chloro-induced contractions were inhibited, although the high concentrations of chloro-induced relaxations were not affected. Taken together, our results indicate that chloro blocks BKs and RyRs, resulting in abolishment of STOCs and occurrence of contraction, the latter will counteract the relaxations induced by high concentrations of chloro.     

Non-Selective Cation Channels Mediate Chloroquine-Induced Relaxation in Precontracted Mouse Airway Smooth Muscle

Bitter tastants can induce relaxation in precontracted airway smooth muscle by activating big-conductance potassium channels (BKs) or by inactivating voltage-dependent L-type Ca²⁺ channels (VDLCCs). In this study, a new pathway for bitter tastant-induced relaxation was defined and investigated. We found nifedipine-insensitive and bitter tastant chloroquine-sensitive relaxation in epithelium-denuded mouse tracheal rings (TRs) precontracted with acetylcholine (ACH). In the presence of nifedipine (10 µM), ACH induced cytosolic Ca²⁺ elevation and cell shortening in single airway smooth muscle cells (ASMCs), and these changes were inhibited by chloroquine. In TRs, ACH triggered a transient contraction under Ca²⁺-free conditions, and, following a restoration of Ca²⁺, a strong contraction occurred, which was inhibited by chloroquine. Moreover, the ACH-activated whole-cell and single channel currents of non-selective cation channels (NSCCs) were blocked by chloroquine. Pyrazole 3 (Pyr3), an inhibitor of transient receptor potential C3 (TRPC3) channels, partially inhibited ACH-induced contraction, intracellular Ca²⁺ elevation, and NSCC currents. These results demonstrate that NSCCs play a role in bitter tastant-induced relaxation in precontracted airway smooth muscle.     

Muscarinic acetylcholine receptor compounds alter net Ca<Superscript>2+</Superscript> flux and contractility in an invertebrate smooth muscle

Responses of a holothurian smooth muscle to a range of muscarinic (M₁ to M₅) acetylcholine receptor (mAChR) agonists and antagonists were surveyed using calcium (Ca²⁺)-selective electrodes and a mechanical recording technique. Most of the mAChR agonists and antagonists tested increased both contractility and net Ca²⁺ efflux, with M₁-specific agents like oxotremorine M being the most potent in their action. To investigate the possible sources of Ca²⁺ used during mAChR activation, agents that disrupt intracellular Ca²⁺ ion sequestration [cyclopiazonic acid (CPA), caffeine, ryanodine], the phosphoinositide signaling pathway [lithium chloride (LiCl)], and L-type Ca²⁺ channels (diltiazem and verapamil) were used to challenge contractions induced by oxotremorine M. These contractions were blocked by treatment with CPA, caffeine, LiCl, and by channel blockers, diltiazem and verapamil, but were unaltered by ryanodine. Our data suggest that this smooth muscle had an M_1,3,5-like receptor that was associated with the phosphoinositide signaling pathway that relied on intracellular Ca²⁺ stores, but secondarily used extracellular Ca²⁺ via the opening of L-type channels.