Role of G proteins in agonist-induced Ca2+ sensitization of tracheal smooth muscle

1998 ◽  
Vol 275 (4) ◽  
pp. L748-L755 ◽  
Author(s):  
Thomas L. Croxton ◽  
Boris Lande ◽  
Carol A. Hirshman
Keyword(s):  
Smooth Muscle ◽  
G Proteins ◽  
Airway Smooth Muscle ◽  
Tracheal Smooth Muscle ◽  
Heterotrimeric G Proteins ◽  
Contractile Responses ◽  
Intracellular Ca ◽  
Increased Sensitivity ◽  
Rho Family

Increased sensitivity to intracellular Ca2+ concentration ([Ca2+]) is an important mechanism for agonist-induced contraction of airway smooth muscle, but the signal transduction pathways involved are uncertain. We studied Ca2+ sensitization with acetylcholine (ACh) and endothelin (ET)-1 in porcine tracheal smooth muscle by measuring contractions at a constant [Ca2+] in strips permeabilized with α-toxin or β-escin. The peptide inhibitor G protein antagonist 2A (GP Ant-2A), which has selectivity for Gq over Gi, inhibited contractile responses to ET-1, ACh, and guanosine 5′- O-(3-thiotriphosphate) (GTPγS), but the proportional inhibition of ACh responses was less than that of ET-1. Pretreatment with pertussis toxin reduced ACh contractions but had no effect on those of ET-1 or GTPγS. Clostridium botulinum C3 exoenzyme, which inactivates Rho family monomeric G proteins, caused similar reductions in contractile responses to ACh, ET-1, and GTPγS. Farnesyltransferase inhibition, which inhibits Ras G proteins, reduced responses to ET-1. We conclude that the heterotrimeric G proteins Gq and Gi both contribute to Ca2+ sensitization by ACh, whereas ET-1 responses involve Gq but not Gi. Both Gq and Gi pathways likely involve Rho family small G proteins. A Ras-mediated pathway also contributes to Ca2+ sensitization by ET-1 in airway smooth muscle.

Halothane Attenuates Calcium Sensitization in Airway Smooth Muscle by Inhibiting G-proteins 

1998 ◽  
Vol 89 (6) ◽  
pp. 1543-1552 ◽  
Author(s):  
Tetsuya Kai ◽  
Keith A. Jones ◽  
David O. Warner
Keyword(s):  
Smooth Muscle ◽  
G Proteins ◽  
Airway Smooth Muscle ◽  
Tracheal Smooth Muscle ◽  
Heterotrimeric G Proteins ◽  
Calcium Sensitization ◽  
Smooth Muscle Preparation ◽  
Guanine Nucleotide Binding ◽  
Nucleotide Binding Proteins ◽  
Guanine Nucleotide Binding Proteins

Background Halothane directly relaxes airway smooth muscle partly by decreasing the Ca2+ sensitivity. In smooth muscle, receptor stimulation is thought to increase Ca2+ sensitivity via a cascade of heterotrimeric and small monomeric guanine nucleotide-binding proteins (G-proteins). Whether this model is applicable in the airway and where halothane acts in this pathway were investigated. Methods A beta-escin-permeabilized canine tracheal smooth muscle preparation was used. Exoenzyme C3 of Clostridium botulinum, which inactivates Rho monomeric G-proteins, was used to evaluate the involvement of this protein in the Ca2+ sensitization pathway. The effects of halothane on different stimulants acting at different levels of signal transduction were compared: acetylcholine on the muscarinic receptor, aluminum fluoride (AIF4-) on heterotrimeric G-proteins, and guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) on all G-proteins. Results Exoenzyme C3 equally attenuated acetylcholine- and AIF4--induced Ca2+ sensitization, suggesting that these pathways are both mediated by Rho. Halothane applied before stimulation equally attenuated acetylcholine- and AIF4--induced Ca2+ sensitization. However, when added after Ca2+ sensitization was established, the effect of halothane was greater during Ca2+ sensitization induced by acetylcholine compared with AIF4-, which, along with the previous result, suggests that halothane may interfere with dissociation of heterotrimeric G-proteins. Halothane applied during GTPgammaS-induced Ca2+ sensitization had no significant effect on force, suggesting that halothane has no effect downstream from monomeric G-proteins. Conclusion Halothane inhibits increases in Ca2+ sensitivity of canine tracheal smooth muscle primarily by interfering with the activation of heterotrimeric G-proteins, probably by inhibiting their dissociation.

Role of cyclic ADP-ribose in the regulation of [Ca2+]i in porcine tracheal smooth muscle

1998 ◽  
Vol 274 (6) ◽  
pp. C1653-C1660 ◽  
Author(s):  
Y. S. Prakash ◽  
Mathur S. Kannan ◽  
Timothy F. Walseth ◽  
Gary C. Sieck
Keyword(s):  
Smooth Muscle ◽  
Second Messenger ◽  
Ruthenium Red ◽  
Tracheal Smooth Muscle ◽  
Cyclic Adp Ribose ◽  
Intracellular Ca ◽  
Subsequent Exposure ◽  
Trisphosphate Receptor ◽  
Dose Dependent Increase

The purpose of the present study was to determine whether cyclic ADP-ribose (cADPR) acts as a second messenger for Ca2+ release through ryanodine receptor (RyR) channels in tracheal smooth muscle (TSM). Freshly dissociated porcine TSM cells were permeabilized with β-escin, and real-time confocal microscopy was used to examine changes in intracellular Ca2+ concentration ([Ca2+]i). cADPR (10 nM–10 μM) induced a dose-dependent increase in [Ca2+]i, which was blocked by the cADPR receptor antagonist 8-amino-cADPR (20 μM) and by the RyR blockers ruthenium red (10 μM) and ryanodine (10 μM), but not by the inositol 1,4,5-trisphosphate receptor blocker heparin (0.5 mg/ml). During steady-state [Ca2+]ioscillations induced by acetylcholine (ACh), addition of 100 nM and 1 μM cADPR increased oscillation frequency and decreased peak-to-trough amplitude. ACh-induced [Ca2+]ioscillations were blocked by 8-amino-cADPR; however, 8-amino-cADPR did not block the [Ca2+]iresponse to a subsequent exposure to caffeine. These results indicate that cADPR acts as a second messenger for Ca2+ release through RyR channels in TSM cells and may be necessary for initiating ACh-induced [Ca2+]ioscillations.

Anesthetics Inhibit Acetylcholine-promoted Guanine Nucleotide Exchange of Heterotrimeric G Proteins of Airway Smooth Muscle

2004 ◽  
Vol 101 (1) ◽  
pp. 120-126 ◽  
Author(s):  
Chie Sakihara ◽  
William J. Perkins ◽  
David O. Warner ◽  
Keith A. Jones
Keyword(s):  
Smooth Muscle ◽  
Muscle Contraction ◽  
G Protein ◽  
Muscarinic Receptor ◽  
G Proteins ◽  
Airway Smooth Muscle ◽  
Smooth Muscle Contraction ◽  
Heterotrimeric G Proteins ◽  
Nucleotide Exchange ◽  
Heterotrimeric G Protein

Background Anesthetics inhibit airway smooth muscle contraction in part by a direct effect on the smooth muscle cell. This study tested the hypothesis that the anesthetics halothane and hexanol, which both relax airway smooth muscle in vitro, inhibit acetylcholine-promoted nucleotide exchange at the alpha subunit of the Gq/11 heterotrimeric G protein (Galphaq/11; i.e., they inhibit muscarinic receptor-Galphaq/11 coupling). Methods The effect of halothane (0.38 +/- 0.02 mm) and hexanol (10 mm) on basal and acetylcholine-stimulated Galphaq/11 guanosine nucleotide exchange was determined in membranes prepared from porcine tracheal smooth muscle. The nonhydrolyzable, radioactive form of guanosine-5'-triphosphate, [S]GTPgammaS, was used as the reporter for Galphaq/11 subunit dissociation from the membrane to soluble fraction, which was immunoprecipitated with rabbit polyclonal anti-Galphaq/11 antiserum. Results Acetylcholine caused a significant time- and concentration-dependent increase in the magnitude of Galphaq/11 nucleotide exchange compared with basal values (i.e., without acetylcholine), reaching a maximal difference at 100 microm (35.9 +/-2.9 vs. 9.8 +/-1.2 fmol/mg protein, respectively). Whereas neither anesthetic had an effect on basal Galphaq/11 nucleotide exchange, both halothane and hexanol significantly inhibited the increase in Galphaq/11 nucleotide exchange produced by 30 microm acetylcholine (by 59% and 68%, respectively). Conclusions Halothane and hexanol interact with the receptor-heterotrimeric G-protein complex in a manner that prevents acetylcholine-promoted exchange of guanosine-5(')-triphosphate for guanosine-5'-diphosphate at Galphaq/11. These data are consistent with the ability of anesthetics to interfere with cellular processes mediated by heterotrimeric G proteins in many cells, including effects on muscarinic receptor-G-protein regulation of airway smooth muscle contraction.

Mechanical strain modulates maximal phosphatidylinositol turnover in airway smooth muscle

1999 ◽  
Vol 277 (5) ◽  
pp. L968-L974 ◽  
Author(s):  
Steven S. An ◽  
Chi-Ming Hai
Keyword(s):  
Smooth Muscle ◽  
G Proteins ◽  
Airway Smooth Muscle ◽  
Mechanical Strain ◽  
Muscle Length ◽  
Heterotrimeric G Proteins ◽  
Maximal Level ◽  
Optimal Length ◽  
Pi Turnover ◽  
Length Dependence

Mechanical strain regulates the maximal level of myosin light chain phosphorylation mediated by muscarinic activation in airway smooth muscle. Accordingly, we tested the hypothesis that mechanical strain regulates maximal phosphatidylinositol (PI) turnover ( V max) coupled to muscarinic receptors in bovine tracheal smooth muscle. We found that PI turnover was not significantly length dependent in unstimulated tissues. However, carbachol-induced PI turnover was linearly dependent on muscle length at both 1 and 100 μM. The observed linear length dependence of PI turnover at maximal carbachol concentration (100 μM) suggests that mechanical strain regulates V max. When carbachol concentration-PI turnover relationships were measured at optimal length and at 20% optimal length, the results could be explained by changes in V max alone. To determine whether the length-dependent step is upstream from heterotrimeric G proteins, we investigated the length dependence of fluoroaluminate-induced PI turnover. The results indicate that fluoroaluminate-induced PI turnover remained significantly length dependent at maximal concentration. These findings together suggest that regulating functional units of G proteins and/or phospholipase C enzymes may be the primary mechanism of mechanosensitive modulation in airway smooth muscle.

Role of platelets in contraction of canine tracheal muscle elicited by PAF in vitro

1988 ◽  
Vol 65 (2) ◽  
pp. 914-920 ◽  
Author(s):  
K. J. Popovich ◽  
G. Sheldon ◽  
M. Mack ◽  
N. M. Munoz ◽  
P. Denberg ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Muscle Contraction ◽  
Airway Smooth Muscle ◽  
Airway Epithelium ◽  
Platelet Activating Factor ◽  
Smooth Muscle Contraction ◽  
Tracheal Smooth Muscle ◽  
Serotonin Antagonist

To elucidate mechanisms of platelet-activating factor (PAF)-induced contraction, we studied the effect of PAF on 203 canine tracheal smooth muscle (TSM) strips from 45 dogs in vitro in the presence and absence of platelets. PAF (10(-11) to 10(-7) M) alone caused no contraction of TSM even in the presence of airway epithelium. In the presence of 2 x 10(5) platelets/microliter, PAF was an extremely potent contractile agonist (threshold 10(-11) M). This response was inhibited by the PAF antagonist, CV-3988 (10(-6) M), and reversed by the serotonin antagonist, methysergide (EC50 = 3.7 +/- 0.79 x 10(-9) M). Neither atropine nor chlorpheniramine (10(-9) to 10(-6) M) attenuated the response to PAF + platelets. In the presence of platelets, 10(-7) M PAF caused an increase in perfusate concentration of serotonin from 0.93 +/- 0.037 x 10(-8) to 1.7 +/- 0.046 x 10(-8) M (P less than 0.001). Tachyphylaxis, previously demonstrated to be irreversible, was shown to be a platelet-dependent phenomenon; contraction could be repeated in the same TSM after addition of fresh platelets. We demonstrate that PAF-induced contraction of canine TSM is caused by the release of cellular intermediates such as serotonin from platelets. We also demonstrate the site of PAF-induced tachyphylaxis in airway smooth muscle contraction.

Caveolae facilitate muscarinic receptor-mediated intracellular Ca2+ mobilization and contraction in airway smooth muscle

2007 ◽  
Vol 293 (6) ◽  
pp. L1406-L1418 ◽  
Author(s):  
Reinoud Gosens ◽  
Gerald L. Stelmack ◽  
Gordon Dueck ◽  
Mark M. Mutawe ◽  
Martha Hinton ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Muscarinic Receptor ◽  
Airway Smooth Muscle ◽  
Isometric Force ◽  
Signaling Proteins ◽  
Muscarinic Agonist ◽  
Caveolin 1 ◽  
Intracellular Ca ◽  
Domain Peptide

Contractile responses of airway smooth muscle (ASM) determine airway resistance in health and disease. Caveolae microdomains in the plasma membrane are marked by caveolin proteins and are abundant in contractile smooth muscle in association with nanospaces involved in Ca2+ homeostasis. Caveolin-1 can modulate localization and activity of signaling proteins, including trimeric G proteins, via a scaffolding domain. We investigated the role of caveolae in contraction and intracellular Ca2+ ([Ca2+]i) mobilization of ASM induced by the physiological muscarinic receptor agonist, acetylcholine (ACh). Human and canine ASM tissues and cells predominantly express caveolin-1. Muscarinic M3 receptors (M3R) and Gαq/11 cofractionate with caveolin-1-rich membranes of ASM tissue. Caveolae disruption with β-cyclodextrin in canine tracheal strips reduced sensitivity but not maximum isometric force induced by ACh. In fura-2-loaded canine and human ASM cells, exposure to methyl-β-cyclodextrin (mβCD) reduced sensitivity but not maximum [Ca2+]i induced by ACh. In contrast, both parameters were reduced for the partial muscarinic agonist, pilocarpine. Fluorescence microscopy revealed that mβCD disrupted the colocalization of caveolae-1 and M3R, but [ N-methyl-3H]scopolamine receptor-binding assay revealed no effect on muscarinic receptor availability or affinity. To dissect the role of caveolin-1 in ACh-induced [Ca2+]i flux, we disrupted its binding to signaling proteins using either a cell-permeable caveolin-1 scaffolding domain peptide mimetic or by small interfering RNA knockdown. Similar to the effects of mβCD, direct targeting of caveolin-1 reduced sensitivity to ACh, but maximum [Ca2+]i mobilization was unaffected. These results indicate caveolae and caveolin-1 facilitate [Ca2+]i mobilization leading to ASM contraction induced by submaximal concentrations of ACh.

Resting calcium influx in airway smooth muscle

2005 ◽  
Vol 83 (8-9) ◽  
pp. 717-723 ◽  
Author(s):  
Luis M Montaño ◽  
Blanca Bazán-Perkins
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Airway Smooth Muscle ◽  
Calcium Influx ◽  
Physiological Role ◽  
Resting Membrane Potential ◽  
Voltage Dependent ◽  
Intracellular Ca ◽  
Influx Pathways

Plasma membrane Ca2+ leak remains the most uncertain of the cellular Ca2+ regulation pathways. During passive Ca2+ influx in non-stimulated smooth muscle cells, basal activity of constitutive Ca2+ channels seems to be involved. In vascular smooth muscle, the 3 following Ca2+ entry pathways contribute to this phenomenon: (i) via voltage-dependent Ca2+ channels, (ii) receptor gated Ca2+ channels, and (iii) store operated Ca2+ channels, although, in airway smooth muscle it seems only 2 passive Ca2+ influx pathways are implicated, one sensitive to SKF 96365 (receptor gated Ca2+ channels) and the other to Ni2+ (store operated Ca2+ channels). Resting Ca2+ entry could provide a sufficient amount of Ca2+ and contribute to resting intracellular Ca2+ concentration ([Ca2+]i), maintenance of the resting membrane potential, myogenic tone, and sarcoplasmic reticulum-Ca2+ refilling. However, further research, especially in airway smooth muscle, is required to better explore the physiological role of this passive Ca2+ influx pathway as it could be involved in airway hyperresponsiveness.Key words: basal Ca2+ entry, constitutive Ca2+ channels, airway and vascular smooth muscle, SKF 96365, Ni2+.

LTD4 induces hyperresponsiveness to histamine in bovine airway smooth muscle: role of SR-ATPase Ca2+ pump and tyrosine kinase

2005 ◽  
Vol 288 (1) ◽  
pp. L84-L92 ◽  
Author(s):  
Verónica Carbajal ◽  
Mario H. Vargas ◽  
Edgar Flores-Soto ◽  
Erasmo Martínez-Cordero ◽  
Blanca Bazán-Perkins ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Tyrosine Kinase ◽  
Airway Smooth Muscle ◽  
Airway Hyperresponsiveness ◽  
Response Curve ◽  
Cyclopiazonic Acid ◽  
Extracellular Signal ◽  
Intracellular Ca ◽  
Concentration Response Curve

Airway hyperresponsiveness is a key feature of asthma, but its mechanisms remain poorly understood. Leukotriene D4 (LTD4) is one of the few molecules capable of producing airway hyperresponsiveness. In this study, LTD4, but not leukotriene C4 (LTC4), produced a leftward displacement of the concentration-response curve to histamine in bovine airway smooth muscle strips. Neither LTC4 nor LTD4 modified the concentration-response curve to carbachol. In simultaneous measurements of intracellular Ca2+ ([Ca2+]i) and contraction, histamine or carbachol produced a transient Ca2+ peak followed by a plateau, along with a contraction. LTD4 increased the histamine-induced transient Ca2+ peak and contraction but did not modify responses to carbachol. Enhanced responses to histamine induced by LTD4 were not modified by staurosporine or chelerythrine but were abolished by genistein. Western blot showed that carbachol, but not histamine, caused intense phosphorylation of extracellular signal-regulated kinase 1/2 and that LTD4 significantly enhanced the phosphorylation induced by histamine, but not by carbachol. L-type Ca2+ channel participation in the hyperresponsiveness to histamine was discarded because LTD4 did not modify the [Ca2+]i changes induced by KCl. In tracheal myocytes, LTD4 enhanced the transient Ca2+ peak induced by histamine (but not by carbachol) and the sarcoplasmic reticulum (SR) Ca2+ refilling. Genistein abolished this last LTD4 effect. Partial blockade of the SR-ATPase Ca2+ pump with cyclopiazonic acid reduced the Ca2+ transient peak induced by histamine but not by carbachol. These results suggested that LTD4 induces hyperresponsiveness to histamine through activation of the tyrosine kinase pathway and an increasing SR-ATPase Ca2+ pump activity. L-type Ca2+ channels seemed not to be involved in this phenomenon.

Cyclopiazonic acid activates a Ca2+-permeable, nonselective cation conductance in porcine and bovine tracheal smooth muscle

2005 ◽  
Vol 99 (5) ◽  
pp. 1759-1768 ◽  
Author(s):  
Peter B. Helli ◽  
Evi Pertens ◽  
Luke J. Janssen
Keyword(s):  
Smooth Muscle ◽  
Patch Clamp ◽  
Airway Smooth Muscle ◽  
Reversal Potential ◽  
Cyclopiazonic Acid ◽  
Membrane Potentials ◽  
Tracheal Smooth Muscle ◽  
Cation Channels ◽  
Cation Conductance ◽  
Intracellular Ca

Capacitative Ca2+ entry has been examined in several tissues and, in some, appears to be mediated by nonselective cation channels collectively referred to as “store-operated” cation channels; however, relatively little is known about the electrophysiological properties of these channels in airway smooth muscle. Consequently we examined the electrophysiological characteristics and changes in intracellular Ca2+ concentration associated with a cyclopiazonic acid (CPA)-evoked current in porcine and bovine airway smooth muscle using patch-clamp and Ca2+-fluorescence techniques. In bovine tracheal myocytes, CPA induced an elevation of intracellular Ca2+ that was dependent on extracellular Ca2+ and was insensitive to nifedipine (an l-type voltage-gated Ca2+ channel inhibitor). Using patch-clamp techniques and conditions that block both K+ and Cl− currents, we found that CPA rapidly activated a membrane conductance ( ICPA) in porcine and bovine tracheal myocytes that exhibits a linear current-voltage relationship with a reversal potential around 0 mV. Replacement of extracellular Na+ resulted in a marked reduction of ICPA at physiological membrane potentials (i.e., −60 mV) that was accompanied by a shift in the reversal potential for ICPA toward more negative membrane potentials. In addition, ICPA was markedly inhibited by 10 μM Gd3+ and La3+ but was largely insensitive to 1 μM nifedipine. We conclude that CPA induces capacitative Ca2+ entry in porcine and bovine tracheal smooth muscle via a Gd3+- and La3+-sensitive, nonselective cation conductance.

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