Role of voltage-gated Ca2+ channels and Ano1 Ca2+-activated Cl- channels in M2 muscarinic receptor-dependent contractions of murine airway smooth muscle.

Abstract

Cholinergic tone is elevated in obstructive lung conditions such as chronic obstructive pulmonary disease (COPD) and asthma, but the cellular mechanisms underlying cholinergic contractions of airway smooth muscle (ASM) are still unclear. Some studies report an important role for L-type Ca²⁺ channels (LTCC) and Ano1 Ca²⁺-activated Cl^- channels (CACC) in these responses, but others dispute their importance. Cholinergic contractions of ASM involve activation of M3Rs, however, stimulation of M2Rs exerts a profound hypersensitization of these responses. Here, we show that M2R-dependent potentiation of cholinergic nerve-evoked contractions of ASM was reversed by the LTCC blocker nifedipine and the Ano1 CACC inhibitors Ani9 and CaCC_inh-A01. Carbachol induced sustained contractions of ASM that were converted into oscillatory contractions when M3Rs were blocked with 4-DAMP. The 4-DAMP-resistant contractions were absent in preparations taken from M2R knockout (KO) mice. The remaining M2R-dependent responses, observed in wild-type (WT) mice, were abolished by nifedipine and Ani9. Inhibition of sarcoplasmic endoplasmic reticulum Ca²⁺ ATPases (SERCA) with thapsigargin increased the amplitude of contractions induced by electrical field stimulation (EFS) and these effects were also reversed by nifedipine and Ani9. Thapsigargin also potentiated contractions of ASM induced by the LTCC activator FPL64176. Therefore, contractions of ASM that involved Ca²⁺ influx via LTCC were enhanced by inhibition of SERCA. Immunocytochemistry experiments revealed prominent SERCA staining around the periphery of ASM cells. These data indicate that M2R-dependent contractions of ASM involve Ano1 CACC and LTCC by a mechanism involving inhibition of buffering of Ca²⁺ influx by SERCA.NEW & NOTEWORTHY The role of L-type Ca²⁺ channels and Ano1 Ca²⁺-activated Cl^- channels in cholinergic contractions of airway smooth muscle is disputed. Here, we show that both channels are involved in M2 muscarinic receptor-dependent contractions of murine airway smooth muscle via inhibition of buffering of Ca²⁺ influx by sarcoplasmic endoplasmic reticulum Ca²⁺ ATPases.