Muscarinic signaling pathway for calcium release and calcium-activated chloride current in smooth muscle.
Abstract: We investigated the muscarinic activation of Ca(2+)-activated Cl- currents [ICl(Ca)] in voltage-clamped equine tracheal myocytes. The threshold of cytosolic free Ca2+ concentration ([Ca2+]i) required for activation of ICl(Ca) was 202 +/- 22 nM, and full activation of the current occurred at 771 +/- 31 nM. Hexahydro-sila-difenidol (M3 antagonist) inhibited the methacholine-induced phasic [Ca2+]i increase and ICl(Ca) in a concentration-dependent manner, whereas methoctramine (M2 antagonist) only slightly attenuated the [Ca2+]i increase and ICl(Ca) (14.8 and 21.4%, respectively), consistent with incomplete selectivity. Dialysis of heparin (10 mg/ml) blocked methacholine-induced [Ca2+]i and ICl(Ca) but had no effect on the caffeine-induced Ca2+ release or ICl(Ca); inositol 1,4,5-trisphosphate (100 microM) induced ICl(Ca) and blocked the methacholine current. Conversely, ruthenium red (50 microM) prevented the caffeine-induced [Ca2+]i release and ICl(Ca) but had no effect on methacholine-induced [Ca2+]i or current. Intracellular dialysis of the calmodulin antagonist N-(6-aminohexyl)-1-naphthalenesulfonamide (W-7, 500 microM) or the Ca2+/calmodulin-dependent protein kinase inhibitor KN93 (5 microM) had no effect on the [Ca2+]i increase or ICl(Ca). Pertussis toxin (0.5 mg/ml) did not affect the increase in [Ca2+]i or ICl(Ca). Dialysis with antibodies directed against the alpha-subunit of Gq/G11 (Gq alpha/ G alpha 11) blocked the methacholine-induced ICl(Ca) in a concentration-dependent manner, whereas anti-G alpha i-1/G alpha 1-2 antibodies (1:35) and anti-G alpha i-3/G(o) alpha antibodies (1:35) were without effect. The results indicate that stimulation of phospholipase C via M3/Gq proteins is the predominant signaling pathway for the activation of ICl(Ca); at high agonist concentrations, Ca(2+)-induced Ca2+ release does not appear to play a prominent role in muscarinic signaling.
Publication Date: 1997-08-01 PubMed ID: 9277348DOI: 10.1152/ajpcell.1997.273.2.C509Google Scholar: Lookup
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- Journal Article
- Research Support
- U.S. Gov't
- P.H.S.
Summary
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This research aims to understand the role of the muscarinic signaling pathway in the activation of calcium-activated chloride currents in smooth muscle cells. The results suggest that stimulation of phospholipase C via M3/Gq proteins is the primary pathway for chloride current activation, and calcium-induced calcium release isn’t as involved as expected in muscarinic signaling when the system is highly stimulated.
Muscarinic Signaling in Tracheal Myocytes
- The researchers studied equine tracheal myocytes, which are smooth muscle cells from the windpipe of horses.
- The signaling pathway involves muscarinic receptors, which activate calcium-activated chloride currents when stimulated. This process is responsible for various important cellular functions like muscle contraction.
Role of Calcium Concentration in Muscarinic Activation
- The requirement for the activation of chloride currents was evaluated to be a specific concentration of free calcium ions in the cytosol (202 +/- 22 nM).
- Full activation of the current occurred when the concentration was higher, at about 771 +/- 31 nM.
Use of Various Antagonists to Test Muscarinic Activation
- The effects of various antagonists like Hexahydro-sila-difenidol and methoctramine on the calcium-ion increase and chloride current were studied. The former inhibited the chloride current while the latter only slightly reduced it. This variation could be due to incomplete selectivity of these antagonists.
- Further tests were conducted using heparin, inositol 1,4,5-trisphosphate, ruthenium red, calmodulin antagonist, calcium/calmodulin-dependent protein kinase inhibitor, and pertussis toxin. These tests offered various results, helping to clarify the roles of different signals in the pathway.
Conclusions on the Role of Phospholipase C
- The study concluded that the main signaling pathway for activating calcium-activated chloride currents involves the stimulation of Phospholipase C via M3/Gq proteins.
- The team also nixed the previously held belief regarding the prominent role calcium-induced calcium release plays in muscarinic signaling, particularly at high concentrations of the activating agent.
Cite This Article
APA
Wang YX, Kotlikoff MI.
(1997).
Muscarinic signaling pathway for calcium release and calcium-activated chloride current in smooth muscle.
Am J Physiol, 273(2 Pt 1), C509-C519.
https://doi.org/10.1152/ajpcell.1997.273.2.C509 Publication
Researcher Affiliations
- Department of Animal Biology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia 19104-6046, USA.
MeSH Terms
- Animals
- Calcium / metabolism
- Calcium / physiology
- Calcium Channels / physiology
- Calcium-Calmodulin-Dependent Protein Kinases / physiology
- Cell Separation
- Chlorides / physiology
- Cytosol / metabolism
- Electric Conductivity
- GTP-Binding Proteins / physiology
- Horses
- Inositol 1,4,5-Trisphosphate Receptors
- Methacholine Chloride / pharmacology
- Muscarine / metabolism
- Muscarinic Agonists / pharmacology
- Muscle, Smooth / cytology
- Muscle, Smooth / metabolism
- Osmolar Concentration
- Pertussis Toxin
- Receptors, Cytoplasmic and Nuclear / physiology
- Receptors, Muscarinic / physiology
- Signal Transduction
- Trachea / cytology
- Trachea / metabolism
- Virulence Factors, Bordetella / pharmacology
Grant Funding
- HL-41084 / NHLBI NIH HHS
- HL-45239 / NHLBI NIH HHS
Citations
This article has been cited 13 times.- Tompkins E, Mimic B, Cuevas-Mora K, Schorsch H, Shah SD, Deshpande DA, Benovic JL, Penn RB, Pera T. PD 102807 Induces M3 mAChR-Dependent GRK-/Arrestin-Biased Signaling in Airway Smooth Muscle Cells.. Am J Respir Cell Mol Biol 2022 Nov;67(5):550-561.
- Tanahashi Y, Komori S, Matsuyama H, Kitazawa T, Unno T. Functions of Muscarinic Receptor Subtypes in Gastrointestinal Smooth Muscle: A Review of Studies with Receptor-Knockout Mice.. Int J Mol Sci 2021 Jan 18;22(2).
- Sghari S, Davies WIL, Gunhaga L. Elucidation of Cellular Mechanisms That Regulate the Sustained Contraction and Relaxation of the Mammalian Iris.. Invest Ophthalmol Vis Sci 2020 Sep 1;61(11):5.
- Yocum GT, Perez-Zoghbi JF, Danielsson J, Kuforiji AS, Zhang Y, Li G, Rashid Roni MS, Kodali R, Stafford DC, Arnold LA, Cook JM, Emala CW Sr. A novel GABA(A) receptor ligand MIDD0301 with limited blood-brain barrier penetration relaxes airway smooth muscle ex vivo and in vivo.. Am J Physiol Lung Cell Mol Physiol 2019 Feb 1;316(2):L385-L390.
- Gallos G, Remy KE, Danielsson J, Funayama H, Fu XW, Chang HY, Yim P, Xu D, Emala CW Sr. Functional expression of the TMEM16 family of calcium-activated chloride channels in airway smooth muscle.. Am J Physiol Lung Cell Mol Physiol 2013 Nov 1;305(9):L625-34.
- Yim PD, Gallos G, Xu D, Zhang Y, Emala CW. Novel expression of a functional glycine receptor chloride channel that attenuates contraction in airway smooth muscle.. FASEB J 2011 May;25(5):1706-17.
- Xiao JH, Zheng YM, Liao B, Wang YX. Functional role of canonical transient receptor potential 1 and canonical transient receptor potential 3 in normal and asthmatic airway smooth muscle cells.. Am J Respir Cell Mol Biol 2010 Jul;43(1):17-25.
- Gallos G, Gleason NR, Virag L, Zhang Y, Mizuta K, Whittington RA, Emala CW. Endogenous gamma-aminobutyric acid modulates tonic guinea pig airway tone and propofol-induced airway smooth muscle relaxation.. Anesthesiology 2009 Apr;110(4):748-58.
- Gallos G, Gleason NR, Zhang Y, Pak SW, Sonett JR, Yang J, Emala CW. Activation of endogenous GABAA channels on airway smooth muscle potentiates isoproterenol-mediated relaxation.. Am J Physiol Lung Cell Mol Physiol 2008 Dec;295(6):L1040-7.
- Montaño LM, Carbajal V, Arreola JL, Barajas-López C, Flores-Soto E, Vargas MH. Acetylcholine and tachykinins involvement in the caffeine-induced biphasic change in intracellular Ca2+ in bovine airway smooth muscle.. Br J Pharmacol 2003 Jul;139(6):1203-11.
- Wang YX, Kotlikoff MI. Signalling pathway for histamine activation of non-selective cation channels in equine tracheal myocytes.. J Physiol 2000 Feb 15;523 Pt 1(Pt 1):131-8.
- Waniishi Y, Inoue R, Morita H, Teramoto N, Abe K, Ito Y. Cyclic GMP-dependent but G-kinase-independent inhibition of Ca2+-dependent Cl- currents by NO donors in cat tracheal smooth muscle.. J Physiol 1998 Sep 15;511 ( Pt 3)(Pt 3):719-31.
- Wang YX, Kotlikoff MI. Inactivation of calcium-activated chloride channels in smooth muscle by calcium/calmodulin-dependent protein kinase.. Proc Natl Acad Sci U S A 1997 Dec 23;94(26):14918-23.
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