Ca 2+ signalling in interstitial cells of Cajal contributes to generation and maintenance of tone in mouse and monkey lower oesophageal sphincters.

The lower oesophageal sphincter (LES) generates tone and prevents reflux of gastric contents. LES smooth muscle cells (SMCs) are relatively depolarised, facilitating activation of Ca v 1.2 channels to sustain contractile tone. We hypothesised that intramuscular interstitial cells of Cajal (ICC-IM), through activation of Ca 2+ -activated Cl - channels (ANO1), set membrane potentials of SMCs favourable for activation of Ca v 1.2 channels. In some gastrointestinal muscles, ANO1 channels in ICC-IM are activated by Ca 2+ transients, but no studies have examined Ca 2+ dynamics in ICC-IM within the LES. Immunohistochemistry and qPCR were used to determine expression of key proteins and genes in ICC-IM and SMCs. These studies revealed that Ano1 and its gene product, ANO1, are expressed in c-Kit + cells (ICC-IM) in mouse and monkey LES clasp muscles. Ca 2+ signalling was imaged in situ, using mice expressing GCaMP6f specifically in ICC (Kit-KI-GCaMP6f). ICC-IM exhibited spontaneous Ca 2+ transients from multiple firing sites. Ca 2+ transients were abolished by cyclopiazonic acid or caffeine but were unaffected by tetracaine or nifedipine. Maintenance of Ca 2+ transients depended on Ca 2+ influx and store reloading, as Ca 2+ transient frequency was reduced in Ca 2+ free solution or by Orai antagonist. Spontaneous tone of LES muscles from mouse and monkey was reduced ∼80% either by Ani9, an ANO1 antagonist or by the Ca v 1.2 channel antagonist nifedipine. Membrane hyperpolarisation occurred in the presence of Ani9. These data suggest that intracellular Ca 2+ activates ANO1 channels in ICC-IM in the LES. Coupling of ICC-IM to SMCs drives depolarisation, activation of Ca v 1.2 channels, Ca 2+ entry and contractile tone. KEY POINTS: The lower oesophageal sphincter (LES) generates contractile tone preventing reflux of gastric contents into the oesophagus. LES smooth muscle cells (SMCs) display depolarised membrane potentials facilitating activation of L-type Ca 2+ channels. Interstitial cells of Cajal (ICC) express Ca 2+ -activated Cl - channels encoded by Ano1 in mouse and monkey LES. Ca 2+ signalling in ICC activates ANO1 currents in ICC. ICC displayed spontaneous Ca 2+ transients in mice from multiple firing sites in each cell and no entrainment of Ca 2+ firing between sites or between cells. Inhibition of ANO1 channels with a specific antagonist caused hyperpolarisation of mouse LES and inhibition of tone in monkey and mouse LES muscles. Our data suggest a novel mechanism for LES tone in which Ca 2+ transient activation of ANO1 channels in ICC generates depolarising inward currents that conduct to SMCs to activate L-type Ca 2+ currents, Ca 2+ entry and contractile tone.