Muscarinic Inhibition of L-type Ca²⁺ Channels in Guinea-Pig Tracheal Smooth Muscle Cells

Abstract

Modulation of L-type Ca²⁺ channels by acetylcholine (ACh) was studied in enzymatically isolated guinea-pig tracheal smooth muscle cells (TSMCs). ACh reversibly inhibited whole-cell L-type Ca²⁺ current measured with Ba²⁺ ions as charge carriers (I_Ba). With pipette solution containing 0.1mM EGTA, 1μM ACh induced transient inhibition of I_Bafollowed by sustained inhibition (67.0±3.7% of the control, n=19). When intracellular Ca²⁺concentration ([Ca²⁺] _i) was fixed at 50nM by BAPTA-Ca²⁺ buffer in the pipette, the transient inhibition was abolished whereas the sustained inhibition (66.0±7.8%, n=6) still occurred, suggesting that the transient inhibition was attributed to inactivation of the channels induced by increase in [Ca²⁺] _i. The sustained inhibition was abolished when [Ca²⁺] _i was fixed at zero. The sustained inhibition of I_Ba by 1μM ACh was observed in the presence of 10μM AF-DX 116, whereas it was not observed in the presence of 1μM4-DAMP. ACh did not inhibit I_Ba, in the presence of 1mM GDP-β-S in the pipette, whereas the drug irreversibly inhibited the current in the presence of 0.1mM GTP-γ-S in the pipette.Pretreatment of TSMCs with pertussis toxin did not altered the effects of ACh. Application of neither 1-oleoyl-2-acetyl-sn-glycerol (1μM) nor phorbol 12-myristate 13-acetate (1μM) reduced T_Ba. These results suggest that the sustained inhibition of I_Ba by ACh is mediated by Ca²⁺ - requiring and protein kinase C-independent mechanisms existing in the downstream of G-protein coupled with M₃ receptors.