Muscarinic signaling pathway for calcium release and calcium-activated chloride current in smooth muscle.
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.
Wang
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YX
Department of Animal Biology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia 19104-6046, USA.
Kotlikoff
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MI
D000818:Animals; D002118:Calcium / Q000378:metabolism / Q000502:physiology*; D015220:Calcium Channels / Q000502:physiology; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000502:physiology; D002469:Cell Separation; D002712:Chlorides / Q000502:physiology*; D003600:Cytosol / Q000378:metabolism; D004553:Electric Conductivity; D019204:GTP-Binding Proteins / Q000502:physiology; D006736:Horses; D053496:Inositol 1,4,5-Trisphosphate Receptors; D016210:Methacholine Chloride / Q000494:pharmacology; D009116:Muscarine / Q000378:metabolism*; D018721:Muscarinic Agonists / Q000494:pharmacology; D009130:Muscle, Smooth / Q000166:cytology / Q000378:metabolism*; D009994:Osmolar Concentration; D037342:Pertussis Toxin; D018160:Receptors, Cytoplasmic and Nuclear / Q000502:physiology; D011976:Receptors, Muscarinic / Q000502:physiology; D015398:Signal Transduction; D014132:Trachea / Q000166:cytology / Q000378:metabolism; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D015220:Calcium Channels; D002712:Chlorides; D053496:Inositol 1,4,5-Trisphosphate Receptors; D018721:Muscarinic Agonists; D018160:Receptors, Cytoplasmic and Nuclear; D011976:Receptors, Muscarinic; D010566:Virulence Factors, Bordetella; D016210:Methacholine Chloride; D009116:Muscarine; D037342:Pertussis Toxin; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D019204:GTP-Binding Proteins; D002118:Calcium