Extraction of membrane cholesterol disrupts caveolae and impairs serotonergic (5-HT2A) and histaminergic (H1) responses in bovine airway smooth muscle: role of Rho-kinase

2009 ◽  
Vol 87 (3) ◽  
pp. 180-195 ◽  
Author(s):  
Bettina Sommer ◽  
Luis M. Montaño ◽  
Verónica Carbajal ◽  
Edgar Flores-Soto ◽  
Alicia Ortega ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Serotonin Receptors ◽  
Rho Kinase ◽  
Membrane Cholesterol ◽  
Histamine Stimulation ◽  
Caveolin 1 ◽  
Intracellular Ca ◽  
Rock Inhibition ◽  
Rock Activity

Some receptors and signaling molecules, such as Rho-kinase (ROCK), localize in caveolae. We asked whether the function of histamine receptors (H1) and 5-hydroxytryptamine (serotonin) receptors (5-HT2A) in bovine tracheal smooth muscle are modified after caveolae disruption and if so, whether the altered ROCK activity plays a role in this modification. Methyl-β-cyclodextrin (MβCD), used to deplete membrane cholesterol, was shown to disrupt caveolae and diminish sustained contractions to histamine (∼80%), 5-HT (100%), α-methyl-5-HT (100%), and KCl (∼30%). Cholesterol-loaded MβCD (CL-MβCD) restored the responses to KCl and partially restored the responses to agonists. ROCK inhibition by Y-27632 diminished contractions to histamine (∼85%) and 5-HT (∼59%). 5-HT or histamine stimulation augmented ROCK activity. These increases were reduced by MβCD and partially reestablished by CL-MβCD. The increase in intracellular Ca2+ that was induced by both agonists was reduced by MβCD. The presence of caveolin-1 (Cav-1), H1, 5-HT2A, and ROCK1 was corroborated by immunoblotting of membrane fractions from sucrose gradients and by confocal microscopy. H1 receptors coimmunoprecipitated with Cav-1 in caveolar and noncaveolar membrane fractions, whereas 5-HT2A receptors appeared to be restricted to noncaveolar membrane fractions. We conclude that caveolar and cholesterol integrity are indispensable for the proper functionality of the H1 and 5-HT2A receptors through their Rho/ROCK signaling.

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.

scholarly journals Differential regulation of muscarinic M2 and M3 receptor signaling in gastrointestinal smooth muscle by caveolin-1

2013 ◽  
Vol 305 (3) ◽  
pp. C334-C347 ◽  
Author(s):  
Sayak Bhattacharya ◽  
Sunila Mahavadi ◽  
Othman Al-Shboul ◽  
Senthilkumar Rajagopal ◽  
John R. Grider ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Muscle Contraction ◽  
Kinase Activity ◽  
Rho Kinase ◽  
Receptor Signaling ◽  
Caveolin 1 ◽  
Pi Hydrolysis ◽  
G Protein Coupled ◽  
Stimulation Of

Caveolae act as scaffolding proteins for several G protein-coupled receptor signaling molecules to regulate their activity. Caveolin-1, the predominant isoform in smooth muscle, drives the formation of caveolae. The precise role of caveolin-1 and caveolae as scaffolds for G protein-coupled receptor signaling and contraction in gastrointestinal muscle is unclear. Thus the aim of this study was to examine the role of caveolin-1 in the regulation of Gq- and Gi-coupled receptor signaling. RT-PCR, Western blot, and radioligand-binding studies demonstrated the selective expression of M2 and M3 receptors in gastric smooth muscle cells. Carbachol (CCh) stimulated phosphatidylinositol (PI) hydrolysis, Rho kinase and zipper-interacting protein (ZIP) kinase activity, induced myosin phosphatase 1 (MYPT1) phosphorylation (at Thr696) and 20-kDa myosin light chain (MLC20) phosphorylation (at Ser19) and muscle contraction, and inhibited cAMP formation. Stimulation of PI hydrolysis, Rho kinase, and ZIP kinase activity, phosphorylation of MYPT1 and MLC20, and muscle contraction in response to CCh were attenuated by methyl β-cyclodextrin (MβCD) or caveolin-1 small interfering RNA (siRNA). Similar inhibition of PI hydrolysis, Rho kinase, and ZIP kinase activity and muscle contraction in response to CCh and gastric emptying in vivo was obtained in caveolin-1-knockout mice compared with wild-type mice. Agonist-induced internalization of M2, but not M3, receptors was blocked by MβCD or caveolin-1 siRNA. Stimulation of PI hydrolysis, Rho kinase, and ZIP kinase activities in response to other Gq-coupled receptor agonists such as histamine and substance P was also attenuated by MβCD or caveolin-1 siRNA. Taken together, these results suggest that caveolin-1 facilitates signaling by Gq-coupled receptors and contributes to enhanced smooth muscle function.

Role of capacitative Ca2+ entry in bronchial contraction and remodeling

2002 ◽  
Vol 92 (4) ◽  
pp. 1594-1602 ◽  
Author(s):  
Michele Sweeney ◽  
Sharon S. McDaniel ◽  
Oleksandr Platoshyn ◽  
Shen Zhang ◽  
Ying Yu ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Transient Receptor Potential ◽  
Bronchial Hyperresponsiveness ◽  
Receptor Potential ◽  
Transient Receptor Potential Channel ◽  
Wall Thickening ◽  
Bronchial Wall ◽  
Intracellular Ca ◽  
Transient Receptor

Asthma is characterized by airway inflammation, bronchial hyperresponsiveness, and airway obstruction by bronchospasm and bronchial wall thickening due to smooth muscle hypertrophy. A rise in cytosolic free Ca2+ concentration ([Ca2+]cyt) may serve as a shared signal transduction element that causes bronchial constriction and bronchial wall thickening in asthma. In this study, we examined whether capacitative Ca2+ entry (CCE) induced by depletion of intracellular Ca2+ stores was involved in agonist-mediated bronchial constriction and bronchial smooth muscle cell (BSMC) proliferation. In isolated bronchial rings, acetylcholine (ACh) induced a transient contraction in the absence of extracellular Ca2+ because of Ca2+ release from intracellular Ca2+ stores. Restoration of extracellular Ca2+in the presence of atropine, an M-receptor blocker, induced a further contraction that was apparently caused by a rise in [Ca2+]cyt due to CCE. In single BSMC, amplitudes of the store depletion-activated currents ( I SOC) and CCE were both enhanced when the cells proliferate, whereas chelation of extracellular Ca2+ with EGTA significantly inhibited the cell growth in the presence of serum. Furthermore, the mRNA expression of TRPC1, a transient receptor potential channel gene, was much greater in proliferating BSMC than in growth-arrested cells. Blockade of the store-operated Ca2+channels by Ni2+ decreased I SOC and CCE and markedly attenuated BSMC proliferation. These results suggest that upregulated TRPC1 expression, increased I SOC, enhanced CCE, and elevated [Ca2+]cyt may play important roles in mediating bronchial constriction and BSMC proliferation.

Functional coupling between the Na+/Ca2+ exchanger and nonselective cation channels during histamine stimulation in guinea pig tracheal smooth muscle

2007 ◽  
Vol 293 (1) ◽  
pp. L191-L198 ◽  
Author(s):  
Paola Algara-Suárez ◽  
Catalina Romero-Méndez ◽  
Tom Chrones ◽  
Sergio Sánchez-Armass ◽  
Ulises Meza ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Guinea Pig ◽  
Force Measurement ◽  
Isometric Force ◽  
Sustained Response ◽  
Functional Coupling ◽  
Channel Activation ◽  
Histamine Stimulation ◽  
Nonselective Cation Channels ◽  
Intracellular Ca

Airway smooth muscle (ASM) contracts partly due to an increase in cytosolic Ca2+. In this work, we found that the contraction caused by histamine depends on external Na+, possibly involving nonselective cationic channels (NSCC) and the Na+/Ca2+ exchanger (NCX). We performed various protocols using isometric force measurement of guinea pig tracheal rings stimulated by histamine. We observed that force reached 53 ± 1% of control during external Na+ substitution by N-methyl-d-glucamine+, whereas substitution by Li+ led to no significant change (91 ± 1%). Preincubation with KB-R7943 decreased the maximal force developed (52.3 ± 5.6%), whereas preincubation with nifedipine did not (89.7 ± 1.8%). Also, application of the nonspecific NCX blocker KB-R7943 and nifedipine on histamine-precontracted tracheal rings reduced force to 1 ± 3%, significantly different from nifedipine alone (49 ± 6%). Moreover, nonspecific NSCC inhibitors SKF-96365 and 2-aminoethyldiphenyl borate reduced force to 1 ± 1% and 19 ± 7%, respectively. Intracellular Ca2+ measurements in isolated ASM cells showed that KB-R7943 and SKF-96365 reduced the peak and sustained response to histamine (0.20 ± 0.1 and 0.19 ± 0.09 for KB-R, 0.43 ± 0.16 and 0.47 ± 0.18 for SKF, expressed as mean of differences). Moreover, Na+-free solution only inhibited the sustained response (0.54 ± 0.25). These data support an important role for NSCC and NCX during histamine stimulation. We speculate that histamine induces Na+ influx through NSCC that promotes the Ca2+ entry mode of NCX and CaV1.2 channel activation, thereby causing contraction.

scholarly journals Expression and functional role of Rho-kinase in rat urinary bladder smooth muscle

2003 ◽  
Vol 138 (5) ◽  
pp. 757-766 ◽  
Author(s):  
Alexandra Wibberley ◽  
Zunxuan Chen ◽  
Erding Hu ◽  
J Paul Hieble ◽  
Timothy D Westfall
Keyword(s):  
Smooth Muscle ◽  
Urinary Bladder ◽  
Functional Role ◽  
Rho Kinase ◽  
Bladder Smooth Muscle ◽  
Rat Urinary Bladder ◽  
Urinary Bladder Smooth Muscle

Atherogenic Activity caused by Excess Membrane Cholesterol in Arterial Smooth Muscle: Role of Calcium Channels

1993 ◽  
pp. 107-115 ◽  
Author(s):  
Thomas N. Tulenko ◽  
R. Preston Mason ◽  
Meng Chen ◽  
Hiromi Tasaki ◽  
Daniel Rock ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Calcium Channels ◽  
Membrane Cholesterol ◽  
Arterial Smooth Muscle

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.

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