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.     

Ca channels and skeletal muscle diseases

When observed under a microscope, skeletal muscle exhibits striations due to the highly organized arrangement of muscle proteins that interact with one another to induce muscle contraction. Muscle contraction requires transient increases in intracellular ‘Ca²⁺’ concentration. In this review, Ca²⁺ channels contributing to the functional integrity of intracellular Ca²⁺-release and extracellular Ca²⁺-entry during skeletal muscle contraction are reviewed in terms of their properties, newly emerging ancillary proteins to them, and their abnormalities related to human skeletal muscle diseases. Finally, the aim of this review is to show the big picture of the correlation among Ca²⁺ channels that participate in the Ca²⁺ homeostasis in skeletal muscle.     

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.     

The role of nitric oxide and l-type calcium channel blocker in the contractility of rabbit ileum in vitro

Nitric oxide (NO) and calcium channel blockers are two agents that can affect gastrointestinal motility. The goal of this work was to study the rabbit intestinal smooth muscle contraction response to (1) sodium nitroprusside (SNP), the NO donor, and its potential mechanism of action, and (2) nifedipine, the l-type Ca²⁺ channel blocker; to clarify the degree of participation by extra- and intracellular Ca²⁺ in smooth muscle contraction. We used standard isometric tension and intracellular micro-electrode recordings. To record the activity of the longitudinal smooth muscle of the ileum, segments of 1.5?cm length of the ileum were suspended vertically in organ baths of Krebs solution. The mechanical activity of the isolated ileal longitudinal muscle was recorded. Different substances were added, and the changes produced on spontaneous contraction were recorded. We found that SNP produced significant decrease, while nitric oxide synthase inhibitor produced significant increase in the amplitude of spontaneous contractions. Both apamin, the Ca²⁺-dependent K⁺ channel blocker, and methylene blue, the inhibitor of soluble guanylate cyclase, alone, partially decreased relaxation induced by SNP. Addition of both methylene blue and apamine together abolished the inhibitory effect produced by SNP on spontaneous contractions. Nifedipine produced significant decrease in the amplitude of spontaneous contractions. In conclusion, in longitudinal muscle of rabbit ileum, calcium channels blocker are potent inhibitors of spontaneous activity. However, both extracellular and intracellular Ca²⁺ participates in the spontaneous contractions. NO also has inhibitory effect on spontaneous activity, and this effect is mediated by cGMP generation system and Ca²⁺-dependent K⁺ channels.     

The role of caveolae and caveolin 1 in calcium handling in pacing and contraction of mouse intestine

In mouse intestine, caveolae and caveolin‐1 (Cav‐1) are present in smooth muscle (responsible for executing contractions) and in interstitial cells of Cajal (ICC; responsible for pacing contractions). We found that a number of calcium handling/dependent molecules are associated with caveolae, including L‐type Ca²⁺ channels, Na⁺‐Ca²⁺ exchanger type 1 (NCX1), plasma membrane Ca²⁺ pumps and neural nitric oxide synthase (nNOS), and that caveolae are close to the peripheral endo‐sarcoplasmic reticulum (ER‐SR). Also we found that this assemblage may account for recycling of calcium from caveolar domains to SR through L‐type Ca ⁺ channels to sustain pacing and contractions. Here we test this hypothesis further comparing pacing and contractions under various conditions in longitudinal muscle of Cav‐1 knockout mice (lacking caveolae) and in their genetic controls. We used a procedure in which pacing frequencies (indicative of functioning of ICC) and contraction amplitudes (indicative of functioning of smooth muscle) were studied in calcium‐free media with 100 mM ethylene glycol tetra‐acetic acid (EGTA). The absence of caveolae in ICC inhibited the ability of ICC to maintain frequencies of contraction in the calcium‐free medium by reducing recycling of calcium from caveolar plasma membrane to SR when the calcium stores were initially full. This recycling to ICC involved primarily L‐type Ca²⁺ channels; i.e. pacing frequencies were enhanced by opening and inhibited by closing these channels. However, when these stores were depleted by block of the sarco/endoplasmic reticulum Ca²⁺‐ATPase (SERCA) pump or calcium release was activated by carbachol, the absence of Cav‐1 or caveolae had little or no effect. The absence of caveolae had little impact on contraction amplitudes, indicative of recycling of calcium to SR in smooth muscle. However, the absence of caveolae slowed the rate of loss of calcium from SR under some conditions in both ICC and smooth muscle, which may reflect the loss of proximity to store operated Ca channels. We found evidence that these channels were associated with Cav‐1. These changes were all consistent with the hypothesis that a reduction of the extracellular calcium associated with caveolae in ICC of the myenteric plexus, the state of L‐type Ca²⁺ channels or an increase in the distance between caveolae and SR affected calcium handling.     

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.     

A mathematical model of airway and pulmonary arteriole smooth muscle

Airway hyperresponsiveness is a major characteristic of asthma and is believed to result from the excessive contraction of airway smooth muscle cells (SMCs). However, the identification of the mechanisms responsible for airway hyperresponsiveness is hindered by our limited understanding of how calcium (Ca²⁺), myosin light chain kinase (MLCK), and myosin light chain phosphatase (MLCP) interact to regulate airway SMC contraction. In this work, we present a modified Hai-Murphy cross-bridge model of SMC contraction that incorporates Ca²⁺ regulation of MLCK and MLCP. A comparative fit of the model simulations to experimental data predicts 1), that airway and arteriole SMC contraction is initiated by fast activation by Ca²⁺ of MLCK; 2), that airway SMC, but not arteriole SMC, is inhibited by a slower activation by Ca²⁺ of MLCP; and 3), that the presence of a contractile agonist inhibits MLCP to enhance the Ca²⁺ sensitivity of airway and arteriole SMCs. The implication of these findings is that murine airway SMCs exploit a Ca²⁺-dependent mechanism to favor a default state of relaxation. The rate of SMC relaxation is determined principally by the rate of release of the latch-bridge state, which is predicted to be faster in airway than in arteriole. In addition, the model also predicts that oscillations in calcium concentration, commonly observed during agonist-induced smooth muscle contraction, cause a significantly greater contraction than an elevated steady calcium concentration.     

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.     

Roles and mechanisms of the CD38/cyclic adenosine diphosphate ribose/Ca2+ signaling pathway Wei W,Graeff R,Yue J简

Mobilization of intracellular Ca²⁺ stores is involved in many diverse cell functions, including: cell proliferation; differentiation; fertilization; muscle contraction; secretion of neurotransmitters, hormones and enzymes; and lymphocyte activation and proliferation. Cyclic adenosine diphosphate ribose (cADPR) is an endogenous Ca²⁺ mobilizing nucleotide present in many cell types and species, from plants to animals. cADPR is formed by ADP-ribosyl cyclases from nicotinamide adenine dinucleotide. The main ADP-ribosyl cyclase in mammals is CD38, a multi-functional enzyme and a type II membrane protein. It has been shown that many extracellular stimuli can induce cADPR production that leads to calcium release or influx, establishing cADPR as a second messenger. cADPR has been linked to a wide variety of cellular processes, but the molecular mechanisms regarding cADPR signaling remain elusive. The aim of this review is to summarize the CD38/cADPR/Ca²⁺ signaling pathway, focusing on the recent advances involving the mechanism and physiological functions of cADPR-mediated Ca²⁺ mobilization.     

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.     

Increase in nuclear calcium in smooth muscle cells exposed to oxidized low density lipoprotein

Vascular smooth muscle cells respond with an increase in intracellular Ca²⁺ within seconds after exposure to oxidized low density lipoprotein (oxLDL). This has been suggested to represent a signaling response that may have implications for gene expression. If so, oxLDL may induce increases in nuclear Ca²⁺ in smooth muscle cells in response to oxLDL. Aortic smooth muscle cells were exposed to 100 μg/ml oxLDL. Large, rapid increases in [Ca²⁺]_i were observed using fluo-3 as an indicator dye to detect intracellular Ca²⁺ on the stage of a confocal micro-scope. This was also confirmed using ratiometric imaging of indo signals. These elevations appeared to be localized to the nuclear region of the cell. DNA staining of the cells confirmed its localization to the nuclear / perinuclear region of the cell. Our data demonstrate that oxLDL induces a nuclear localized elevation in Ca²⁺i that may have important implications for nuclear function.     

Folium Sennae and emodin reverse airway smooth muscle contraction

The objective of this project was to find a bronchodilatory compound from herbs and clarify the mechanism. We found that the ethanol extract of Folium Sennae (EEFS) can relax airway smooth muscle (ASM). EEFS inhibited ASM contraction, induced by acetylcholine, in mouse tracheal rings and lung slices. High‐performance liquid chromatography assay showed that EEFS contained emodin. Emodin had a similar reversal action. Acetylcholine‐evoked contraction was also partially reduced by nifedipine (a selective inhibitor of L‐type voltage‐dependent Ca²⁺ channels, LVDCCs), YM‐58483 (a selective inhibitor of store‐operated Ca²⁺ entry, SOCE), as well as Y‐27632 (an inhibitor of Rho‐associated protein kinase). In addition, LVDCC‐ and SOCE‐mediated currents and cytosolic Ca²⁺ elevations were inhibited by emodin. Emodin reversed acetylcholine‐caused increases in phosphorylation of myosin phosphatase target subunit 1. Furthermore, emodin, in vivo, inhibited acetylcholine‐induced respiratory system resistance in mice. These results indicate that EEFS‐induced relaxation results from emodin inhibiting LVDCC, SOCE, and Ca²⁺ sensitization. These findings suggest that Folium Sennae and emodin may be new sources of bronchodilators.     

Effects of elevated cytosolic calcium on ACh-induced swine tracheal smooth muscle contraction

Increased intracellular calcium concentration ([Ca²⁺]_i) is required for smooth muscle contraction. In tracheal and other tonic smooth muscles, contraction and elevated [Ca²⁺]_i are maintained as long as an agonist is present. To evaluate the physiological role of steady-state increases in Ca²⁺ on tension maintenance, [Ca²⁺]_i was elevated using ionomycin, a Ca²⁺ ionophore or charybdotoxin, a large-conductance calcium-activated potassium channel (K_Ca) blocker prior to or during exposure of tracheal smooth muscle strips to Ach (10^–9 to 10^–4 M). Ionomycin (5 µM) in resting muscles induced increases in [Ca²⁺]_i to 500±230 nM and small increases in force of 2.6±2.3 N/cm². This tension is only 10% of the maximal tension induced by ACh. Charybdotoxin had no effect on [Ca²⁺]_i or tension in resting muscle. After pretreatment of muscle with ionomycin, the concentration-response relationship for ACh-induced changes in tension shifted to the left (EC₅₀=0.07±0.05 µM ionomycin; 0.17±0.07 µM, control, p<0.05). When applied to the muscles during steady-state responses to submaximal concentrations of ACh, both ionomycin and charybdotoxin induced further increases in tension. The same magnitude increase in tension occurs after ionomycin and charybdotoxin treatment, even though the increase in [Ca²⁺]_i induced by charybdotoxin is much smaller than that induced by ionomycin. We conclude that the resting muscle is much less sensitive to elevation of [Ca²⁺]_i when compared to muscles stimulated with ACh. Steady-state [Ca²⁺]_i limits tension development induced by submaximal concentrations of ACh. The activity of K_Ca moderates the response of the muscle to ACh at concentrations less than 1 µM.     

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.     

The role of STIM1 and SOCE in smooth muscle contractility

Contraction is a central feature for skeletal, cardiac and smooth muscle; this unique feature is largely dependent on calcium (Ca²⁺) signaling and therefore maintenance of internal Ca²⁺ stores. Stromal interaction molecule 1 (STIM1) is a single-pass transmembrane protein that functions as a Ca²⁺ sensor for the activation store-operated calcium channels (SOCCs) on the plasma membrane in response to depleted internal sarco(endo)plasmic (S/ER) reticulum Ca²⁺ stores. STIM1 was initially characterized in non-excitable cells; however, evidence from both animal models and human mutations suggests a role for STIM1 in modulating Ca²⁺ homeostasis in excitable tissues as well. STIM1-dependent SOCE is particularly important in tissues undergoing sustained contraction, leading us to believe STIM1 may play a role in smooth muscle contraction. To date, the role of STIM1 in smooth muscle is unknown. In this review, we provide a brief overview of the role of STIM1-dependent SOCE in striated muscle and build off that knowledge to investigate whether STIM1 contributes to smooth muscle contractility. We conclude by discussing the translational implications of targeting STIM1 in the treatment of smooth muscle disorders.     

Mathematical Modelling of Ca2+ Oscillations in Airway Smooth Muscle Cells

The action of different agonists such as acetylcholine on the membrane of airway smooth muscle cells may induce cytosolic Ca²⁺ oscillations which can be a part of the Ca²⁺ signalling pathway, eventually leading to cell contraction. The aim of the present study is to present a mathematical model of the possible effect of the initial Ca²⁺ distribution within the cell on the form and frequency of induced Ca²⁺ oscillations. It takes into account intracellular Ca²⁺ stores such as sarcoplasmic reticulum and cytosolic proteins as well as Ca²⁺ exchange across the plasma membrane. We are able to demonstrate a closer agreement of model predictions with observed Ca²⁺ traces for a significantly wider range of parameter values, as was previously reported. We show also that the total cellular Ca²⁺ content is an important system parameter especially because of the content in sarcoplasmic reticulum. At a total Ca²⁺ increase of about 20%, the oscillation frequency increases by 25%; also, damped oscillations become sustained. Cases are indicated in which such a situation could occur.     

Dietary Selenium Influences Calcium Release and Activation of MLCK in Uterine Smooth Muscle of Rats

We sought to elucidate the effects of different concentrations of dietary selenium on calcium ion release, MLCK levels, and muscle contraction in the uterine smooth muscle of rats. The selenium (Se) content of blood and of uterine smooth muscle tissues was detected by fluorescence spectrophotometry. Ca²⁺ content was measured by atomic absorption spectroscopy. Calmodulin (CaM) and MLCK RNA and protein levels were analyzed by quantitative real-time polymerase chain reaction and Western blot, respectively. Dietary Se intake increased the Se levels in the blood and in uterine smooth muscle tissues and increased the Ca²⁺ concentration in uterine smooth muscle tissues. The addition of Se also promoted CaM expression and enhanced MLCK activation in uterine smooth muscle tissues. In conclusion, Ca²⁺, CaM, and MLCK were regulated by Se in uterine smooth muscle; Se plays a major role in regulating smooth muscle contraction in the uterus.     

A Ca2+‐binding protein with numerous roles and uses: parvalbumin in molecular biology and physiology

Parvalbumins (PVs) are acidic, intracellular Ca²⁺‐binding proteins of low molecular weight. They are associated with several Ca²⁺‐mediated cellular activities and physiological processes. It has been suggested that PV might function as a “Ca²⁺ shuttle” transporting Ca²⁺ from troponin‐C (TnC) to the sarcoplasmic reticulum (SR) Ca²⁺ pump during muscle relaxation. Thus, PV may contribute to the performance of rapid, phasic movements by accelerating the contraction–relaxation cycle of fast‐twitch muscle fibers. Interestingly, PVs promote the generation of power stroke in fish by speeding up the rate of relaxation and thus provide impetus to attain maximal sustainable speeds. However, immunological monitoring of diverse tissues demonstrated that PVs are also present in non‐muscle cells. The axoplasmic transport and various intracellular secretory mechanisms including the endocrine secretions seem to be controlled by the Ca²⁺ regulation machinery. Any defect in the Ca²⁺ handling apparatus may cause several clinical problems; for instance, PV deficiency alters the neuronal activity, a key mechanism leading to epileptic seizures. Moreover, atypical relaxation of the heart results in diastolic dysfunction, which is a major cause of heart failure predominantly among the aged people. PV may offer a unique potential to correct defective relaxation in energetically compromised failing hearts through PV gene transfer. Consequently, PV gene transfer may present a new therapeutic approach to correct cellular disturbances in Ca²⁺ signaling pathways of diseased organs. Hence, PVs appear to be amazingly useful candidate proteins regulating a variety of cellular functions through action on Ca²⁺ flux management.     

Calcium and ionophore A23187 stimulates deposition of extracellular matrix and acetylcholinesterase release in cultured myotubes

Summary Calcium (Ca²⁺) and calcium-transporting ionophores stimulate protein secretion in many cellular systems. We demonstrate here that increases in intracellular calcium concentration induce a time- and concentration-dependent deposition of extracellular matrix and an increase in acetylcholinesterase secretion. Scanning and transmission electron-microscopy revealed that treatment with the calcium ionophore A23187, or high extracellular Ca²⁺ levels (5 mM to 15 mM) produce significant deposits of extracellular matrix around the myotubes, as well as a marked increase in the acetylcholinesterase reaction-product. Blocking muscle contraction was not necessary for the induction of AChE secretory activity. Sucrose density-gradients of media conditioned by muscle cells revealed 3 separate acetylcholinesterase molecular forms. However, incubation with A23187 increased only the 4.5 S and the 7.2 S molecular forms, whereas the 12.0 S form showed no significant differences from controls. Polyacrylamide gel electrophoresis, and autoradiography using [³H]diisopropyl fluorophosphate revealed a broad band at 65000 daltons. This band was broader than for controls when medium was obtained from A23187-treated cells. Our results show that increasing intracellular Ca²⁺ concentration induces marked deposition of extracellular matrix and increased acetylcholinesterase secretion, with an apparent selectivity for the monomeric and dimeric acetylcholinesterase molecular forms.     

Ca2+ Sparks Act as Potent Regulators of Excitation-Contraction Coupling in Airway Smooth Muscle

Ca²⁺ sparks are short lived and localized Ca²⁺ transients resulting from the opening of ryanodine receptors in sarcoplasmic reticulum. These events relax certain types of smooth muscle by activating big conductance Ca²⁺-activated K⁺ channels to produce spontaneous transient outward currents (STOCs) and the resultant closure of voltage-dependent Ca²⁺ channels. But in many smooth muscles from a variety of organs, Ca²⁺ sparks can additionally activate Ca²⁺-activated Cl⁻ channels to generate spontaneous transient inward current (STICs). To date, the physiological roles of Ca²⁺ sparks in this latter group of smooth muscle remain elusive. Here, we show that in airway smooth muscle, Ca²⁺ sparks under physiological conditions, activating STOCs and STICs, induce biphasic membrane potential transients (BiMPTs), leading to membrane potential oscillations. Paradoxically, BiMPTs stabilize the membrane potential by clamping it within a negative range and prevent the generation of action potentials. Moreover, blocking either Ca²⁺ sparks or hyperpolarization components of BiMPTs activates voltage-dependent Ca²⁺ channels, resulting in an increase in global [Ca²⁺]_i and cell contraction. Therefore, Ca²⁺ sparks in smooth muscle presenting both STICs and STOCs act as a stabilizer of membrane potential, and altering the balance can profoundly alter the status of excitability and contractility. These results reveal a novel mechanism underlying the control of excitability and contractility in smooth muscle.